Chemical ligation dependent probe amplification (clpa)

ABSTRACT

The present invention provides compositions, apparatuses and methods for detecting one or more nucleic acid targets present in a sample. Methods of the invention include utilizing two or more oligonucleotide probes that reversibly bind a target nucleic acid in close proximity to each other and possess complementary reactive ligation moieties. When such probes have bound to the target in the proper orientation, they are able to undergo a spontaneous chemical ligation reaction that yields a ligated oligonucleotide product. In one aspect, the ligation product is of variable length that correlates with a particular target. Following chemical ligation, the probes may be amplified and detected by capillary electrophoresis or microarray analysis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.12/798,108 filed Mar. 29, 2010, which claims priority from U.S.Provisional Application No. 61/165,839, filed Apr. 1, 2009, thedisclosures of which are incorporated by this reference as though setforth fully herein.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

The sequence listing contained in the file named 068433-5002-US01_ST25”,created on May 21, 2018, and having a size of 8.77 kilobytes, has beensubmitted electronically herewith via EFS-Web, and the contents of thetxt file are hereby incorporated by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to compositions and methods for detecting nucleicacids in a sample using chemical ligation.

BACKGROUND OF THE INVENTION

This invention relates to compositions, apparatus and methods fordetecting one or more nucleic acid targets present in a sample. Thedetection of specific nucleic acids is an important tool for diagnosticmedicine and molecular biology research.

Gene probe assays currently play roles in identifying infectiousorganisms such as bacteria and viruses, in probing the expression ofnormal and mutant genes and identifying genes associated with disease orinjury, such as oncogenes, in typing tissue for compatibility precedingtissue transplantation, in matching tissue or blood samples for forensicmedicine, for responding to emergency response situations like a nuclearincident or pandemic flu outbreak, in determining disease prognosis orcausation, and for exploring homology among genes from differentspecies.

Ideally, a gene probe assay should be sensitive, specific and easilyautomatable (for a review, see Nickerson, Current Opinion inBiotechnology (1993) 4:48-51.) The requirement for sensitivity (i.e. lowdetection limits) has been greatly alleviated by the development of thepolymerase chain reaction (PCR) and other amplification technologieswhich allow researchers to exponentially amplify a specific nucleic acidsequence before analysis (for a review, see Abramson et al., CurrentOpinion in Biotechnology, (1993) 4:41-47). For example, multiplex PCRamplification of SNP loci with subsequent hybridization tooligonucleotide arrays has been shown to be an accurate and reliablemethod of simultaneously genotyping hundreds of SNPs (see Wang et al.,Science, (1998) 280:1077; see also Schafer et al., Nature Biotechnology,(1989)16:33-39).

Specificity also remains a problem in many currently available geneprobe assays. The extent of molecular complementarity between probe andtarget defines the specificity of the interaction. Variations incomposition and concentrations of probes, targets and salts in thehybridization reaction as well as the reaction temperature, and lengthof the probe may all alter the specificity of the probe/targetinteraction.

It may be possible under some circumstances to distinguish targets withperfect complementarity from targets with mismatches, although this isgenerally very difficult using traditional technology, since smallvariations in the reaction conditions will alter the hybridization.Newer techniques with the necessary specificity for mismatch detectioninclude probe digestion assays in which mismatches create sites forprobe cleavage, and DNA ligation assays where single point mismatchesprevent ligation.

A variety of enzymatic and non-enzymatic methods are available fordetecting sequence variations. Examples of enzyme based methods includeInvader™, oligonucleotide ligation assay (OLA) single base extensionmethods, allelic PCR, and competitive probe analysis (e.g. competitivesequencing by hybridization). Enzymatic DNA ligation reactions are wellknown in the art (Landegren, Bioessays (1993) 15(11):761-5; Pritchard etal., Nucleic Acids Res. (1997) 25(17):3403-7; Wu et al., Genomics,(1989) 4(4):560-9) and have been used extensively in SNP detection,enzymatic amplification reactions and DNA repair.

A number of non-enzymatic or template mediated chemical ligation methodshave been developed that can be used to detect sequence variations.These include chemical ligation methods that utilize coupling reagents,such as N-cyanoimidazole, cyanogen bromide, and1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride. SeeMetelev, V. G., et al., Nucleosides & Nucleotides (1999) 18:2711;Luebke, K. J., and Dervan, P. B. J. Am. Chem. Soc. (1989) 111:8733; andShabarova, Z. A., et al., Nucleic Acids Research (1991)19:4247, each ofwhich is incorporated herein by reference in its entirety.

Kool (U.S. Pat. No 7,033,753), which is incorporated herein by referencein its entirety describes the use of chemical ligation and fluorescenceresonance energy transfer (FRET) to detect genetic polymorphisms. Thereadout in this process is based on the solution phase change influorescent intensity.

Terbrueggen (U.S. Patent application 60/746,897) which is incorporatedherein by reference in its entirety describes the use of chemicalligation methods, compositions and reagents for the detection of nucleicacids via microarray detection.

Other chemical ligation methods react a 5′-tosylate or 5′-iodo groupwith a 3′-phosphorothioate group, resulting in a DNA structure with asulfur replacing one of the bridging phosphodiester oxygen atoms. SeeGryanov, S. M., and Letsinger, R. L., Nucleic Acids Research (1993)21:1403; Xu, Y. and Kool, E. T. Tetrahedron Letters (1997) 38:5595; andXu, Y. and Kool, E. T., Nucleic Acids Research (1999) 27:875, each ofwhich is herein incorporated by reference in its entirety.

Some of the advantages of using non-enzymatic approaches for nucleicacid target detection include lower sensitivity to non-natural DNAanalog structures, ability to use RNA target sequences, lower cost andgreater robustness under varied conditions. Letsinger et al (U.S. Pat.No. 5,780,613, herein incorporated by reference in its entirety) havepreviously described an irreversible, nonenzymatic, covalentautoligation of adjacent, template-bound oligonucleotides wherein oneoligonucleotide has a 5′ displaceable group and the otheroligonucleotide has a 3′ thiophosphoryl group.

PCT applications WO 95/15971, PCT/US96/09769, PCT/US97/09739, PCTUS99/01705, WO96/40712 and WO98/20162, all of which are expresslyincorporated herein by reference in their entirety, describe novelcompositions comprising nucleic acids containing electron transfermoieties, including electrodes, which allow for novel detection methodsof nucleic acid hybridization.

One technology that has gained increased prominence involves the use ofDNA arrays (Marshall et al., Nat Biotechnol. (1998) 16(1):27-31),especially for applications involving simultaneous measurement ofnumerous nucleic acid targets. DNA arrays are most often used for geneexpression monitoring where the relative concentration of 1 to 100,000nucleic acids targets (mRNA) is measured simultaneously. DNA arrays aresmall devices in which nucleic acid anchor probes are attached to asurface in a pattern that is distinct and known at the time ofmanufacture (Marshall et al., Nat Biotechnol. (1998) 16(1):27-31) or canbe accurately deciphered at a later time such as is the case for beadarrays (Steemers et al., Nat Biotechnol. (2000) 18(1):91-4; and Yang etal., Genome Res. (2001) 11(11):1888-98.). After a series of upstreamprocessing steps, the sample of interest is brought into contact withthe DNA array, the nucleic acid targets in the sample hybridize toanchor oligonucleotides on the surface, and the identity and oftenconcentration of the target nucleic acids in the sample are determined.

Many of the nucleic acid detection methods in current use havecharacteristics and/or limitations that hinder their broadapplicability. For example, in the case of DNA microarrays, prior tobringing a sample into contact with the microarray, there are usually aseries of processing steps that must be performed on the sample. Whilethese steps vary depending upon the manufacturer of the array and/or thetechnology that is used to read the array (fluorescence,electrochemistry, chemiluminescence, magnetoresistance, cantileverdeflection, surface plasmon resonance), these processing steps usuallyfall into some general categories: Nucleic acid isolation andpurification, enzymatic amplification, detectable label incorporation,and clean up post-amplification. Other common steps are sampleconcentration, amplified target fragmentation so as to reduce theaverage size of the nucleic acid target, and exonuclease digestion toconvert PCR amplified targets to a single stranded species.

The requirement of many upstream processing steps prior to contactingthe DNA array with the sample can significantly increase the time andcost of detecting a nucleic acid target(s) by these methods. It can alsohave significant implications on the quality of the data obtained. Forinstance, some amplification procedures are very sensitive to targetdegradation and perform poorly if the input nucleic acid material is notwell preserved (Foss et al., Diagn Mol Pathol. (1994) 3(3):148-55).Technologies that can eliminate or reduce the number and/or complexityof the upstream processing steps could significantly reduce the cost andimprove the quality of results obtained from a DNA array test. Onemethod for reducing upstream processing steps involves using ligationreactions to increase signal strength and improve specificity.

There remains a need for methods and compositions for efficient andspecific nucleic acid detection. Accordingly, the present inventionprovides methods and compositions for non-enzymatic chemical ligationreactions which provides very rapid target detection and greatlysimplified processes of detecting and measuring nucleic acid targets.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the invention relates to a method comprisingproviding a ligation substrate comprising a target nucleic acid sequencecomprising at least a first target domain and a second target domain,and a first and second ligation probe. The ligation probes may comprisea stuffer sequence of variable length and/or sequence. The firstligation probe comprises a first probe domain substantiallycomplementary to the first target domain, and a 5′-ligation moiety. Thesecond ligation probe comprises a second probe domain substantiallycomplementary to the second target domain, and a 3′ ligation moiety.Optionally, the first target domain and the second target domain areseparated by at least one nucleotide. Optionally, at least one of thefirst and said second ligation probes comprises an anchor sequenceand/or a label, including a label probe binding sequence. The first andsecond ligation probes are ligated in the absence of exogeneously addedligase enzyme to form a ligation product. The ligated product mayoptionally be captured on a substrate comprising a capture probesubstantially complementary to said anchor sequence and detected. Theligation product may be amplified and detected by capillaryelectrophoresis, microarray analysis, or any other suitable method.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of one embodiment of CLPA-CE assay.

FIG. 2. Schematic representation of one embodiment of CLPA-MDM assay.

FIG. 3. Schematic representation showing one embodiment of the 2-probeand the 3-probe CLPA reaction.

FIG. 4. Schematic Representation of a DNA synthesis resin that can beused to manufacture DNA with a 3′-DABSYL leaving group

FIG. 5. Schematic Representation on the process flow for one embodimentof the CLPA-CE assay

FIG. 6. Schematic chart showing probe design for CLPA assay in which isincorporated a size-variant stuffer sequence.

FIG. 7. Electrophoretic separation profile on sample analyzed byCLPA-CE.

FIG. 8. Linear relationship between target concentration and peak heightin CLPA-CE analysis.

DETAILED DESCRIPTION OF THE INVENTION

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can also be used. Suchconventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry 3rdEd., W. H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W. H. Freeman Pub., New York, N.Y., all ofwhich are hereby incorporated in their entirety by reference for allpurposes. Furthermore, all references cited in this application areherein incorporated in their entirety by reference for all purposes.

Overview

The invention provides compositions, apparatus and methods for thedetection of one or more nucleic acid targets in a sample including DNAand RNA targets. Moreover, the sample need not be purified. Indeed, oneaspect of the invention relates to analyzing impure samples includingbody samples such as, but not limited to, whole blood. The inventionprovides methods utilizing two or more oligonucleotide probes thatreversibly bind a target nucleic acid in close proximity to each otherand possess complementary reactive ligation moieties (it should benoted, as is further described herein, that the reactive moieties arereferred to herein as “ligation moieties”). When the probes have boundthe target in the proper orientation, they are able to undergo aspontaneous chemical ligation reaction that yields a ligatedoligonucleotide product. Following ligation, a new product is generatedthat can be amplified by an enzymatic or chemical reaction. In thepreferred embodiment, the chemical ligation reaction joins two probesthat have PCR primer sites on them, e.g. universal PCR primers.Additionally, in one embodiment of the invention, one or both ligationprobes contain a stuffer sequence, or variable spacer sequence, which isdesigned to have differing lengths for each probe set (i.e. each targetsequence) thereby resulting in a ligation product having atarget-specific length. Following ligation a defined lengtholigonucleotide can now be exponentially amplified by PCR. In accordancewith one aspect of the invention, the probes can possess detectablelabels (fluorescent labels, electrochemical labels, magnetic beads,nanoparticles, biotin) to aid in the identification, purification,quantification or detection of the ligated oligonucleotide product. Theprobes may also optionally include in their structure: anchoringoligonucleotide sequences designed for subsequent capture on a solidsupport (microarrays, microbeads, nanoparticles), molecule handles thatpromote the concentration or manipulation of the ligated product(magnetic particles, oligonucleotide coding sequences), and promotersequences to facilitate subsequent secondary amplification of theligated product via an enzyme like a DNA or RNA polymerase. The ligationreactions of the invention proceed rapidly, are specific for thetarget(s) of interest, and can produce multiple copies of the ligatedproduct for each target(s), resulting in an amplification (sometimesreferred to herein as “product turnover”) of the detectable signal. Theligation reactions of the invention do not require the presence ofexogeneously added ligases, nor additional enzymes, although somesecondary reactions may rely on the use of enzymes such as polymerases,as described below. Ligation chemistries can be chosen from many of thepreviously described chemical moieties. Preferred chemistries are onesthat can be easily incorporated into routine manufacture techniques, arestable during storage, and demonstrate a large preference for targetspecific ligation when incorporated into a properly designed ligationprobe set. Additionally, for embodiments which involve subsequentamplification by an enzyme, ligation chemistries and probe designs(including unnatural nucleotide analogs) that result in a ligationproduct that can be efficiently processed by an enzyme are preferred.Amplification of the target may also include turnover of the ligationproduct, in which the ligation product has a lower or comparableaffinity for the template or target nucleic acid than do the separateligation probes. Thus, upon ligation of the hybridized probes, theligation product is released from the target, freeing the target toserve as a template for a new ligation reaction.

In one embodiment, the ligation reactions of the invention includetransfer reactions. In this embodiment, the probes hybridize to thetarget sequence, but rather than oligonucleotide probes being ligatedtogether to form a ligation product, a nucleic acid-directed transfer ofa molecular entity (including reporter molecules such as fluorophores,quenchers, etc) from one oligonucleotide probe to other occurs. Thistransfer reaction is analogous to a ligation reaction, however insteadof joining of two or more probes, one of the probes is ligated to thetransfer molecule and the other probe is the “leaving group” of thechemical reaction. We use the term “transfer” reaction so as todistinguish between the different nature of the resulting final product.Importantly, similar to the ligation reaction, the transfer reaction isfacilitated by the proximal binding of the transfer probes onto anucleic acid target, such that significant signal is detected only ifthe probes have hybridized to the target nucleic acid in close enoughproximity to one another (e.g., at adjacent sites) for the transferreaction to take place.

Samples

Accordingly, in one aspect the present invention provides compositionsand methods for detecting the presence or absence of target sequences insamples. As will be appreciated by those in the art, the sample solutionmay comprise any number of things, including, but not limited to, bodilyfluids (including, but not limited to, blood, urine, serum, lymph,saliva, anal and vaginal secretions, perspiration and semen, ofvirtually any organism, with mammalian samples being preferred and humansamples being particularly preferred); environmental samples (including,but not limited to, air, agricultural, water and soil samples); plantmaterials; biological warfare agent samples; research samples (forexample, the sample may be the product of an amplification reaction, forexample general amplification of genomic DNA); purified samples, such aspurified genomic DNA, RNA, proteins, etc.; raw samples (bacteria, virus,genomic DNA, etc.); as will be appreciated by those in the art,virtually any experimental manipulation may have been done on thesample. Some embodiments utilize siRNA and microRNA as target sequences(Zhang et al., J Cell Physiol. (2007) 210(2):279-89; Osada et al.,Carcinogenesis. (2007) 28(1):2-12; and Mattes et al., Am J Respir CellMol Biol. (2007) 36(1):8-12, each of which is incorporated herein byreference in its entirety).

Some embodiments of the invention utilize nucleic acid samples fromstored (e.g. frozen and/or archived) or fresh tissues. Paraffin-embeddedsamples are of particular use in many embodiments, as these samples canbe very useful, due to the presence of additional data associated withthe samples, such as diagnosis and prognosis. Fixed andparaffin-embedded tissue samples as described herein refers to storableor archival tissue samples. Most patient-derived pathological samplesare routinely fixed and paraffin-embedded to allow for histologicalanalysis and subsequent archival storage. Such samples are often notuseful for traditional methods of nucleic acid detection, because suchstudies require a high integrity of the nucleic acid sample so that anaccurate measure of nucleic acid expression can be made. Often, geneexpression studies in paraffin-embedded samples are limited toqualitative monitoring by using immunohistochemical staining to monitorprotein expression levels.

Methods and compositions of the present invention are useful indetection of nucleic acids from paraffin-embedded samples, because theprocess of fixing and embedding in paraffin often results in degradationof the samples' nucleic acids. The present invention is able to amplifyand detect even degraded samples, such as those found inparaffin-embedded samples.

A number of techniques exist for the purification of nucleic acids fromfixed paraffin-embedded samples as described in WO 2007/133703 theentire contents of which is herein incorporated by reference.

In a preferred embodiment, the target analytes are nucleic acids. By“nucleic acid” or “oligonucleotide” or grammatical equivalents hereinmeans at least two nucleotides covalently linked together. A nucleicacid of the present invention will generally contain phosphodiesterbonds (for example in the case of the target sequences), although insome cases, as outlined below, nucleic acid analogs are included thatmay have alternate backbones (particularly for use with the ligationprobes), comprising, for example, phosphoramide (Beaucage et al.,Tetrahedron (1993) 49(10):1925 and references therein; Letsinger, J.Org. Chem. (1970) 35:3800; Sprinzl et al., Eur. J. Biochem. (1977)81:579; Letsinger et al., Nucl. Acids Res. (1986) 14:3487; Sawai et al,Chem. Lett. (1984) 805; Letsinger et al., J. Am. Chem. Soc. (1988)110:4470; and Pauwels et al., Chemica Scripta (1986) 26:141),phosphorothioate (Mag et al., Nucleic Acids Res. (1991) 19:1437; andU.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem.Soc. (1989) 111:2321, O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem. Soc. (1992)114:1895; Meier et al., Chem. Int. Ed. Engl. (1992)31:1008; Nielsen, Nature, (1993) 365:566; Carlsson et al., Nature (1996)380:207, all of which are incorporated herein by reference in theirentirety). Other analog nucleic acids include those with bicyclicstructures including locked nucleic acids, Koshkin et al., J. Am. Chem.Soc. (1998) 120:13252 3); positive backbones (Denpcy et al., Proc. Natl.Acad. Sci. USA (1995) 92:6097; non-ionic backbones (U.S. Pat. Nos.5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi etal., Angew. Chem. Intl. Ed. English (1991) 30:423; Letsinger et al., J.Am. Chem. Soc. (1988) 110:4470; Letsinger et al., Nucleoside &Nucleotide (1994) 13:1597; Chapters 2 and 3, ASC Symposium Series 580,Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic &Medicinal Chem. Lett. (1994) 4:395 ; Jeffs et al., J. Biomolecular NMR(1994) 34:17; Xu et al., Tetrahedron Lett. (1996) 37:743) and non-ribosebackbones, including those described in U.S. Pat. Nos. 5,235,033 and5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Ed. Y. S.Sanghui and P. Dan Cook. Nucleic acids containing one or morecarbocyclic sugars are also included within the definition of nucleicacids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Severalnucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997page 35. All of these references are herein expressly incorporated byreference. These modifications of the ribose-phosphate backbone may bedone to facilitate the addition of labels or other moieties, to increaseor decrease the stability and half-life of such molecules inphysiological environments, etc.

As will be appreciated by those in the art, all of these nucleic acidanalogs may find use in the present invention. In addition, mixtures ofnaturally occurring nucleic acids and analogs can be made; for example,at the site of a ligation moiety, an analog structure may be used.Alternatively, mixtures of different nucleic acid analogs, and mixturesof naturally occurring nucleic acids and analogs may be made.

Nucleic acid analogue may include, for example, peptide nucleic acid(PNA, WO 92/20702, incorporated herein by reference in its entirety) andLocked Nucleic Acid (LNA, Koshkin A A et al. Tetrahedron (1998)54:3607-3630., Koshkin A A et al. J. Am. Chem. Soc. (1998)120:13252-13253., Wahlestedt C et al. PNAS (2000) 97:5633-5638, each ofwhich is incorporated herein by reference in its entirety). In someapplications analogue backbones of this type may exhibit improvedhybridization kinetics, improved thermal stability and improvedsensitivity to mismatch sequences.

The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequences. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includingnaturally occurring nucleobases (uracil, adenine, thymine, cytosine,guanine) and non-naturally occurring nucleobases (inosine, xathaninehypoxathanine, isocytosine, isoguanine, 5-methylcytosine,pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine,N9-(2-amino-6-chloropurine), N9-(2,6-diaminopurine), hypoxanthine,N9-(7-deaza-guanine), N9-(7-deaza-8-aza-guanine) andN8-(7-deaza-8-aza-adenine). 5-propynyl-uracil, 2-thio-5-propynyl-uracil)etc. As used herein, the term “nucleobase” includes both “nucleosides”and “nucleotides”, and monomers of nucleic acid analogs. Thus, forexample, the individual units of a peptide nucleic acid, each containinga base, are referred to herein as a nucleobase.

In one aspect, ligation probes of the invention are any polymericspecies that is capable of interacting with a nucleic acid target(s) ina sequence specific manner and possess chemical moieties allowing theprobes to undergo a spontaneous chemical ligation reaction with anotherpolymeric species possessing complementary chemical moieties. In oneembodiment, the oligonucleotide probes can be DNA, RNA, PNA, LNA,modified versions of the aforementioned and/or any hybrids of the same(e.g. DNA/RNA hybrids, DNA/LNA hybrids, DNA/PNA hybrids). In a preferredembodiment, the oligonucleotide probes are DNA or RNA oligonucleotides.

Nucleic acid samples (e.g. target sequences) that do not exist in asingle-stranded state in the region of the target sequence(s) aregenerally rendered single-stranded in such region(s) prior to detectionor hybridization. Generally, nucleic acid samples can be renderedsingle-stranded in the region of the target sequence using heatdenaturation. For polynucleotides obtained via amplification, methodssuitable for generating single-stranded amplification products arepreferred. Non-limiting examples of amplification processes suitable forgenerating single-stranded amplification product polynucleotidesinclude, but are not limited to, T7 RNA polymerase run-offtranscription, RCA, Asymmetric PCR (Bachmann et al., Nucleic Acid Res.(1990) 18:1309), and Asynchronous PCR (WO 01/94638). Commonly knownmethods for rendering regions of double-stranded polynucleotides singlestranded, such as the use of PNA openers (U.S. Pat. No. 6,265,166), mayalso be used to generate single-stranded target sequences on apolynucleotide.

In one aspect, the invention provides methods of detecting targetsequences. By “target sequence” or “target nucleic acid” or grammaticalequivalents herein means a nucleic acid sequence on a single strand ofnucleic acid. The target sequence may be a portion of a gene, aregulatory sequence, genomic DNA, cDNA, RNA including mRNA, MicroRNA andrRNA, or others. As is outlined herein, the target sequence may be atarget sequence from a sample, or a secondary target such as a productof an amplification reaction, etc. It may be any length, with theunderstanding that longer sequences are more specific. As will beappreciated by those in the art, the complementary target sequence maytake many forms. For example, it may be contained within a largernucleic acid sequence, i.e. all or part of a gene or mRNA, a restrictionfragment of a plasmid or genomic DNA, among others.

In some embodiments, the target sequence is comprised of different typesof target domain. For example, a first target domain of the sampletarget sequence may hybridize to a first ligation probe, and a secondtarget domain in the target sequence may hybridize to a second ligationprobe. Other target domains may hybridize to a capture probe on asubstrate such as an array, or a label probe, etc..

The target domains may be adjacent or separated as indicated, as is morefully described below. In some cases, when detection is based onligation and the application requires amplification of signal, theligation probes may utilize linkers and be separated by one or morenucleobases of the target sequence to confer hybridization instabilityon the ligated product. In other applications, for example in singlenucleotide polymorphism (SNP) detection, or in transfer reactions, theligation probes may hybridize to adjacent nucleobases of the targetsequence. Unless specified, the terms “first” and “second” are not meantto confer an orientation of the sequences with respect to the 5′-3′orientation of the target sequence. For example, assuming a 5′-3′orientation of the complementary target sequence, the first targetdomain may be located either 5′ to the second domain, or 3′ to thesecond domain. For ease of reference and not to be limiting, thesedomains are sometimes referred to as “upstream” and “downstream”, withthe normal convention being the target sequence being displayed in a 5′to 3′ orientation

The probes are designed such that when the probes bind to a part of thetarget polynucleotide in close spatial proximity, a chemical ligationreaction occurs between the probes. In general, the probes comprisechemically reactive moieties (herein generally referred to as “ligationmoieties”) and bind to the target polynucleotide in a particularorientation, such that the chemically reactive moieties come into closespatial proximity, thus resulting in a spontaneous ligation reaction.

Probe Components

In one embodiment, the invention provides sets of ligation probes,usually a first and a second ligation probe, although as is describedherein some embodiments utilize more than two. In addition, as notedherein, in some cases a transfer reaction is done rather than ligation;“ligation probes” includes “transfer probes”. Each ligation probecomprises a nucleic acid portion, sometimes referred to herein as a“probe domain” that is substantially complementary to one of the targetdomains. Probes of the present invention are designed to becomplementary to a target sequence such that hybridization of the targetsequence and the probes of the present invention occurs. As outlinedherein, this complementarity need not be perfect; there may be anynumber of base pair mismatches which will interfere with hybridizationbetween the target sequence and the probes of the present invention.However, if the number of mutations is so great that no hybridizationcan occur under even the least stringent of hybridization conditions,the sequence is not a complementary sequence. Thus, by “substantiallycomplementary” herein is meant that the probes are sufficientlycomplementary to the target sequences to hybridize under normal reactionconditions. “Identical” sequences are those that over the length of theshorter sequence of nucleobases, perfect complementarity exists.

In one aspect of the invention, the length of the probe is designed tovary with the length of the target sequence, the specificity required,the reaction (e.g. ligation or transfer) and the hybridization and washconditions. Generally, in this aspect ligation probes range from about 5to about 150 nucleobases, with from about 15 to about 100 beingpreferred and from about 25 to about 75 being especially preferred. Ingeneral, these lengths apply equally to ligation and transfer probes.

In another embodiment of the invention, referred to herein as “CLPA-CE”which is described more fully below, probe length is designed to varyfor each target of interest thereby generating ligation products thatcan be identified and analyzed based on length variance.

A variety of hybridization conditions may be used in the presentinvention, including high, moderate and low stringency conditions; seefor example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2dEdition, 1989, and Ausubel, et al, Short Protocols in Molecular Biology,herein incorporated by reference. The hybridization conditions may alsovary when a non-ionic backbone, e.g. PNA is used, as is known in theart.

Ligation Moieties

In addition to ligation domains, the ligation probes of the inventionhave ligation moieties. Accordingly, in one aspect, the inventionrelates to methods of chemical ligation that include the binding of atleast a first and a second ligation probe to the target nucleic acid toform a “ligation substrate” under conditions such that the ligationmoieties of the first and second ligation probes are able tospontaneously react, ligating the probes together, in the absence ofexogenous ligase; that is, no exogenous ligase is added to the reaction.In the case of the transfer reaction, this may be referred to as eithera “ligation substrate” or a “transfer substrate”. By “ligationsubstrate” herein is meant a substrate for chemical ligation comprisingat least one target nucleic acid sequence and two or more ligationprobes. Similarly, included within the definition of “ligationsubstrate” is a “transfer substrate”, comprising at least one targetnucleic acid sequence and two or more transfer probes.

In some embodiments of the invention, for example when additionalspecificity is desired, more than two ligation probes can be used. Inthis embodiment, the “middle” ligation probe(s) can also be adjacent orseparated by one or more nucleobases of the target sequence. In apreferred embodiment, the ligation reaction does not require thepresence of a ligase enzyme and occurs spontaneously between the boundprobes in the absence of any addition (e.g. exogeneous) ligase.

Oligonucleotide probes of the invention are designed to be specific forthe polynucleotide target. These probes bind to the target in closespatial proximity to each other and are oriented in such a manner thatthe chemically reactive moieties are in close spatial proximity. In oneaspect, two or more probes are designed to bind near adjacent sites on atarget polynucleotide. In a preferred embodiment, two probes bind to thetarget such that the ligation moiety at the 5′ end of oneoligonucleotide probe is able to interact with the ligation moiety atthe 3′ end of the other probe.

Chemical ligation can, under appropriate conditions, occur spontaneouslywithout the addition of any additional activating reagents or stimuli.Alternatively, “activating” agents or external stimuli can be used topromote the chemical ligation reaction. Examples of activating agentsinclude, without limitation, carbodiimide, cyanogen bromide (BrCN),imidazole, 1-methylimidazole/carbodiimide/cystamine, N-cyanoimidazole,dithiothreitol (DTT), tris(2-carboxyethyl)phosphine (TCEP) and otherreducing agents as well as external stimuli like ultraviolet light, heatand/or pressure changes.

As is outlined herein, the ligation moieties of the invention may take avariety of configurations, depending on a number of factors. Most of thechemistries depicted herein are used in phosphoramidite reactions thatgenerally progress in a 3′ to 5′ direction. That is, the resin containschemistry allowing attachment of phosphoramidites at the 5′ end of themolecule. However, as is known in the art, phosphoramidites can be usedto progress in the 5′ to 3′ direction; thus, the invention includesmoieties with opposite orientation to those outlined herein.

Each set of ligation probes (or transfer probes) contains a set of afirst ligation moiety and a second ligation moiety. The identificationof these ligation moiety pairs depends on the chemistry of the ligationto be used. In addition, as described herein, linkers (including but notlimited to destabilization linkers) may be present between the probedomain and the ligation moiety of one or both ligation probes. Ingeneral, for ease of discussion, the description herein may use theterms “upstream” and “downstream” ligation probes, although this is notmeant to be limiting.

Halo Leaving Group Chemistry

In one embodiment of the invention, the chemistry is based on 5′ halogenleaving group technology such as is generally described in Gryanov, S.M., and Letsinger, R. L., (1993) Nucleic Acids Research, 21:1403; Xu, Y.and Kool, E. T. (1997) Tetrahedron Letters, 38:5595; Xu, Y. and Kool, E.T., (1999) Nucleic Acids Research, 27:875; Arar et al., (1995), BioConj.Chem., 6:573; Kool, E. T. et. al, (2001) Nature Biotechnol 19:148; Kool,E. T. et. al., (1995) Nucleic Acids Res, 23 (17):3547; Letsinger et al.,U.S. Pat. No. 5,476,930; Shouten et al., U.S. Pat. No. 6,955,901;Andersen et al., U.S. Pat. No. 7,153,658, all of which are expresslyincorporated by reference herein. In this embodiment, the first ligationprobe includes at its 5′ end a nucleoside having a 5′ leaving group, andthe second ligation probe includes at its 3′ end a nucleoside having 3′nucleophilic group such as a 3′ thiophosphoryl. The 5′ leaving group caninclude many common leaving groups know to those skilled in the artincluding, for example the halo-species (I, Br, Cl) and groups such asthose described by Abe and Kool, J. Am. Chem. Soc. (2004)126:13980-13986, which is incorporated herein by reference in itsentirety. In a more preferred embodiment of this aspect of theinvention, the first ligation probe has a 5′ leaving group attachedthrough a flexible linker and a downstream oligonucleotide which has a3′ thiophosphoryl group. This configuration leads to a significantincrease in the rate of reaction and results in multiple copies ofligated product being produced for every target.

The “upstream” oligonucleotide, defined in relation to the 5′ to 3′direction of the polynucleotide template as the oligonucleotide thatbinds on the “upstream” side (i.e., the left, or 5′ side) of thetemplate includes, as its 5′ end, a 5′-leaving group. Any leaving groupcapable of participating in an S_(N)2 reaction involving sulfur,selenium, or tellurium as the nucleophile can be utilized. The leavinggroup is an atom or group attached to carbon such that on nucleophilicattack of the carbon atom by the nucleophile (sulfur, selenium ortellurium) of the modified phosphoryl group, the leaving group leaves asan anion. Suitable leaving groups include, but are not limited to ahalide, such as iodide, bromide or chloride, a tosylate,benzenesulfonate or p-nitrophenylester, as well as RSO₃ where R isphenyl or phenyl substituted with one to five atoms or groups comprisingF, Cl, Br, I, alkyl (C1 to C6), nitro, cyano, sulfonyl and carbonyl, orR is alkyl with one to six carbons. The leaving group is preferably aniodide, and the nucleoside at the 5′ end of the upstream oligonucleotideis, in the case of DNA, a 5′-deoxy-5′-iodo-2′-deoxynucleoside. Examplesof suitable 5′-deoxy-5′-iodo-2′-deoxynucleosides include, but are notlimited to, 5′-deoxy-5′-iodothymidine (5′-I-T),5′-deoxy-5′-iodo-2′-deoxycytidine (5′-I-dC),5′-deoxy-5′-iodo-2′-deoxyadenosine (5′-I-dA),5′-deoxy-5′-iodo-3-deaza-2′-deoxyadenosine (5′-I-3-deaza-dA),5′-deoxy-5′-iodo-2′-deoxyguanosine (5′-I-dG) and5′-deoxy-5′-iodo-3-deaza-2′-deoxyguanosine (5′-I-3-deaza-dG), and thephosphoroamidite derivatives thereof (see FIG. 2). In the case of RNAoligonucleotides, analogous examples of suitable5′-deoxy-5′-iodonucleosides include, but are not limited to,5′-deoxy-5′-iodouracil (5′-I-U), 5′-deoxy-5′-iodocytidine (5′-I-C),5′-deoxy-5′-iodoadenosine (5′-I-A), 5′-deoxy-5′-iodo-3-deazaadenosine(5′-I-3-deaza-A), 5′-deoxy-5′-iodoguanosine (5′-I-G) and5′-deoxy-5′-iodo-3-deazaguanosine (5′-I-3-deaza-G), and thephosphoroamidite derivatives thereofIn a preferred embodiment, anupstream ligation probe contains 2′-deoxyribonucleotides except that themodified nucleotide on the 5′ end, which comprises the 5′ leaving group,is a ribonucleotide. This embodiment of the upstream nucleotide isadvantageous because the bond between the penultimate2′-deoxyribonucleotide and the terminal 5′ ribonucleotide is susceptibleto cleavage using base. This allows for potential reuse of anoligonucleotide probe that is, for example, bound to a solid support, asdescribed in more detail below. In reference to the CLPA assay, which isdescribed more fully below, the 5′ leaving group of the “upstream” probeis most preferably DAB SYL.

The “downstream” oligonucleotide, which binds to the polynucleotidetemplate “downstream” of, i.e., 3′ to, the upstream oligonucleotide,includes, as its 3′ end, a nucleoside having linked to its 3′ hydroxyl aphosphorothioate group (i.e., a “3′-phosphorothioate group”), aphosphoroselenoate group (i.e., a “3′-phosphoroselenoate group), or aphosphorotelluroate group (i.e., a “3′-phosphorotelluroate group”). Thechemistries used for autoligation are thus sulfur-mediated,selenium-mediated, or tellurium mediated. Self-ligation yields aligation product containing a 5′ bridging phosphorothioester(—O—P(O)(O.sup.-)-S-), phosphoroselenoester (—O—P(O)(O.sup.-)-Se-) orphosphorotelluroester (—O—P(O)(O.sup.-)-Te-), as dictated by the groupcomprising the 3′ end of the downstream oligonucleotide. Thisnon-natural, achiral bridging diester is positioned between two adjacentnucleotides and takes the place of a naturally occurring 5′ bridgingphosphodiester. Surprisingly, the selenium-mediated ligation is 3 to 4times faster than the sulfur-mediated ligation, and theselenium-containing ligation product was very stable, despite the lowerbond strength of the Se—P bond. Further, the bridgingphosphoroselenoester, as well as the bridging phosphorotelluroester, areexpected to be cleavable selectively by silver or mercuric ions undervery mild conditions (see Mag et al., Nucleic Acids Res. (1991) 19:14371441).

In one embodiment, a downstream oligonucleotide contains2′-deoxyribonucleotides except that the modified nucleotide on the 3′end, which comprises the 3′ phosphorothioate, phosphoroselenoate, orphosphorotelluroate, is a ribonucleotide. This embodiment of theupstream nucleotide is advantageous because the bond between thepenultimate 2′-deoxyribonucleotide and the terminal ribonucleotide issusceptible to cleavage using base, allowing for potential reuse of anoligonucleotide probe that is, for example, bound to a solid support. Inreference to the CLPA assay, as described more fully below, the“downstream” probe most preferably includes at its 3′ end3′-phosphorothioate.

It should be noted that the “upstream” and “downstream” oligonucleotidescan, optionally, constitute the two ends of a single oligonucleotide, inwhich event ligation yields a circular ligation product. The bindingregions on the 5′ and 3′ ends of the linear precursor oligonucleotidemust be linked by a number of intervening nucleotides sufficient toallow binding of the 5′ and 3′ binding regions to the polynucleotidetarget.

Compositions provided by the invention include a5′-deoxy-5-'iodo-2′-deoxynucleoside, for example a5′-deoxy-5′-iodothymidine (5′-I-T), 5′-deoxy-5′-iodo-2′-deoxycytidine(5′-I-dC), 5′-deoxy-5′-iodo-2′-deoxyadenosine (5′-I-dA),5′-deoxy-5′-iodo-3-deaza-2′-deoxyadenosine (5′-I-3-deaza-dA),5′-deoxy-5′-iodo-2′-deoxyguanosine (5′-I-dG) and5′-deoxy-5′-iodo-3-deaza-2′-deoxyguanosine (5′-I-3-deaza-dG), and thephosphoroamidite derivatives thereof, as well as an oligonucleotidecomprising, as its 5′ end, a 5′-deoxy-5′-iodo-2′-deoxynucleoside of theinvention. Compositions provided by the invention further include a5′-deoxy-5′-iodonucleoside such as 5′-deoxy-5′-iodouracil (5′-I-U),5′-deoxy-5′-iodocytidine (5′-I-C), 5′-deoxy-5′-iodoadenosine (5′-1-A),5′-deoxy-5′-iodo-3-deazaadenosine (5′-I-3-deaza-A),5′-deoxy-5′-iodoguanosine (5′-I-G) and 5′-deoxy-5′-iodo-3-deazaguanosine(5′-I-3-deaza-G), and the phosphoroamidite derivatives thereof, as wellas an oligonucleotide comprising, as its 5′ end, a5′-deoxy-5′-iodonucleoside of the invention. Also included in theinvention is a nucleoside comprising a 3′-phosphoroselenoate group or a3′-phosphorotelluroate group, and an oligonucleotide comprising as its3′ end a nucleoside comprising a 3′-phosphoroselenoate group or a3′-phosphorotelluroate group. Oligonucleotides containing either or bothof these classes of modified nucleosides are also included in theinvention, as are methods of making the various nucleosides andoligonucleotides. Oligonucleotides that are modified at either or bothof the 5′ or 3′ ends in accordance with the invention optionally, butneed not, include a detectable label, preferably a radiolabel, afluorescence energy donor or acceptor group, an excimer label, or anycombination thereof.

In addition, in some cases, substituent groups may also be protectinggroups (sometimes referred to herein as “PG”). Suitable protectinggroups will depend on the atom to be protected and the conditions towhich the moiety will be exposed. A wide variety of protecting groupsare known; for example, DMT is frequently used as a protecting group inphosphoramidite chemistry (as depicted in the figures; however, DMT maybe replaced by other protecting groups in these embodiments. A widevariety of protecting groups are suitable; see for example, Greene'sProtective Groups in Organic Synthesis, herein incorporated by referencefor protecting groups and associated chemistry.

By “alkyl group” or grammatical equivalents herein is meant a straightor branched chain alkyl group, with straight chain alkyl groups beingpreferred. If branched, it may be branched at one or more positions, andunless specified, at any position. The alkyl group may range from about1 to about 30 carbon atoms (C1-C30), with a preferred embodimentutilizing from about 1 to about 20 carbon atoms (C1-C20), with about C1through about C12 to about C15 being preferred, and C1 to C5 beingparticularly preferred, although in some embodiments the alkyl group maybe much larger. Also included within the definition of an alkyl groupare cycloalkyl groups such as C5 and C6 rings, and heterocyclic ringswith nitrogen, oxygen, sulfur or phosphorus. Alkyl also includesheteroalkyl, with heteroatoms of sulfur, oxygen, nitrogen, and siliconebeing preferred. Alkyl includes substituted alkyl groups. By“substituted alkyl group” herein is meant an alkyl group furthercomprising one or more substitution moieties “R”, as defined above.

By “amino groups” or grammatical equivalents herein is meant NH₂, —NHRand —NR₂ groups, with R being as defined herein. In some embodiments,for example in the case of the peptide ligation reactions, primary andsecondary amines find particular use, with primary amines generallyshowing faster reaction rates.

By “nitro group” herein is meant an —NO₂ group.

By “sulfur containing moieties” herein is meant compounds containingsulfur atoms, including but not limited to, thia-, thio- andsulfo-compounds, thiols (—SH and —SR), and sulfides (—RSR—). Aparticular type of sulfur containing moiety is a thioester (—(CO)—S—),usually found as a substituted thioester (—(CO)—SR). By “phosphoruscontaining moieties” herein is meant compounds containing phosphorus,including, but not limited to, phosphines and phosphates. By “siliconcontaining moieties” herein is meant compounds containing silicon.

By “ether” herein is meant an —O—R group. Preferred ethers includealkoxy groups, with —O—(CH₂)₂ CH₃ and —O—(CH₂)₄ CH₃ being preferred.

By “ester” herein is meant a —COOR group.

By “halogen” herein is meant bromine, iodine, chlorine, or fluorine.Preferred substituted alkyls are partially or fully halogenated alkylssuch as CF₃, etc.

By “aldehyde” herein is meant —RCOH groups.

By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

By “amido” herein is meant —RCONH— or RCONR— groups.

By “ethylene glycol” herein is meant a —(O—CH₂—CH₂)_(n)— group, althougheach carbon atom of the ethylene group may also be singly or doublysubstituted, i.e. —(—CR₂—CR₂)_(n)—, with R as described above. Ethyleneglycol derivatives with other heteroatoms in place of oxygen (i.e.—(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or with substitution groups) arealso preferred.

Additionally, in some embodiments, the R group may be a functionalgroup, including quenchers, destabilization moieties and fluorophores(as defined below). Fluorophores of particular use in this embodimentinclude, but are not limited to Fluorescein and its derivatizes, TAMRA(Tetramethyl-6-carboxyrhodamine), Alexa dyes, and Cyanine dyes (e.g. Cy3and Cy5).

Quencher moieties or molecules are known in the art, and are generallyaromatic, multiring compounds that can deactivate the excited state ofanother molecule. Fluorophore-quencher pairs are well known in the art.Suitable quencher moieties include, but are not limited to Dabcyl(Dimethylamini(azobenzene) sulfonyl) Dabcyl(Dimethylamino(azobenzene)carbonyl), Eclipse Quenchers (Glen ResearchCatalog) and blackhole Quenchers (BHQ-1, BHQ-2 and BHQ-3) from BiosearchTechnologies.

Suitable destabilization moieties are discussed below and include, butare not limited to molecule entities that result in a decrease in theoverall binding energy of an oligonucleotide to its target site.Potential examples include, but are not limited to alkyl chains, chargedcomplexes, and ring structures.

Nucleophile Ligation Moieties

In this embodiment, the other ligation probe comprises a ligation moietycomprising a nucleophile such as an amine. Ligation moieties comprisingboth a thiol and an amine find particular use in certain reactions. Ingeneral, the nucleophile ligation moieites can include a wide variety ofpotential amino, thiol compounds as long as the nucleophile ligationmoiety contains a thiol group that is proximal to a primary or secondaryamino and the relative positioning is such that at least a 5 or 6 memberring transition state can be achieve during the S to N acyl shift.

Accordingly, nucleophile ligation molecules that comprise 1, 2 or 1, 3amine thiol groups find particular use. Primary amines find use in someembodiments when reaction time is important, as the reaction time isgenerally faster for primary than secondary amines, although secondaryamines find use in acyl transferase reactions that contribute todestabilization as discussed below. The carbons between the amino andthiol groups can be substituted with non-hydrogen R groups, althoughgenerally only one non-hydrogen R group per carbon is utilized.Additionally, adjacent R groups (depicted as R′ and R″ in Figure *CC)may be joined together to form cyclic structures, including substitutedand unsubstituted cycloalkyl and aryl groups, including heterocycloalkyland heteroaryl and the substituted and unsubstituted derivativesthereof. In the case where a 1,2 amino thiol group is used and adjacentR groups are attached, it is generally preferred that the adjacent Rgroups form cycloalkyl groups (including heterocycloalkyl andsubstituted derivatives thereof) rather than aryl groups.

In this embodiment, for the generation of the 4 sigma bond contractionof the chain for destabilization, the replacement ligation moiety relieson an acyl transferase reaction.

Linkers

In many embodiments, linkers (sometimes shown herein as “L” or“-(linker)_(n)—), (where n is zero or one) may optionally be included ata variety of positions within the ligation probe(s). Suitable linkersinclude alkyl and aryl groups, including heteroalkyl and heteroaryl, andsubstituted derivatives of these. In some instances, for example whenNative Peptide Ligation reactions are done, the linkers may be aminoacid based and/or contain amide linkages. As described herein, somelinkers allow the ligation probes to be separated by one or morenucleobases, forming abasic sites within the ligation product, whichserve as destabilization moieties, as described below.

Destabilization Moieties

In accordance with one aspect of the invention, it is desirable toproduce multiple copies of ligated product for each target moleculewithout the aid of an enzyme. One way to achieve this goal involves theligated product disassociating from the target following the chemicalligation reaction to allow a new probe set to bind to the target. Toincrease ligation product turnover, probe designs, instrumentation, andchemical ligation reaction chemistries that increase productdisassociation from the target molecule are desirable.

Previous work has shown one way to achieve product disassociation andincrease product turnover is to “heat cycle” the reaction mixture. Heatcycling is the process of varying the temperature of a reaction so as tofacilitate a desired outcome. Most often heat cycling takes the form ofbriefly raising the temperature of the reaction mixture so that thereaction temperature is above the melting temperature of the ligatedproduct for a brief period of time causing the product to disassociatefrom the target. Upon cooling, a new set of probes is able to bind thetarget, and undergo another ligation reaction. This heat cyclingprocedure has been practiced extensively for enzymatic reactions likePCR.

While heat cycling is one way to achieve product turnover, it ispossible to design probes such that there is significant productturnover without heat cycling. Probe designs and ligation chemistriesthat help to lower the melting temperature of the ligated productincrease product turnover by decreasing product inhibition of thereaction cycle.

Accordingly, in one aspect, the probes are designed to include elements(e.g. destabilization moieties), which, upon ligation of the probes,serve to destabilize the hybridization of the ligation product to thetarget sequence. As a result, the ligated substrate disassociates afterligation, resulting in a turnover of the ligation product, e.g. theligation product comprising the two ligation probes dehybridizes fromthe target sequence, freeing the target sequence for hybridization toanother probe set.

In addition, increasing the concentration of the free (e.g.unhybridized) ligation probes can also help drive the equilibriumtowards release of the ligation product (or transfer product) from thetarget sequence. Accordingly, some embodiments of the invention useconcentrations of probes that are 1,000,000 fold higher than that of thetarget while in other embodiments the probes are 10,000 to 100 foldhigher than that of the target. As will be appreciated by those skilledin the art, increasing the concentration of free probes can be used byitself or with any embodiment outlined herein to achieve productturnover (e.g. amplification). While increasing the probe concentrationcan result in increased product turnover, it can also lead tosignificant off target reactions such as probe hydrolysis and non-targetmediated ligation.

In one aspect, probe elements include structures which lower the meltingtemperature of the ligated product. In some embodiments, probe elementsare designed to hybridize to non-adjacent target nucleobases, e.g. thereis a “gap” between the two hybridized but unligated probes. In general,this is done by using one or two linkers between the probe domain andthe ligation moiety. That is, there may be a linker between the firstprobe domain and the first ligation moiety, one between the second probedomain and the second ligation moiety, or both. In some embodiments, thegap comprises a single nucleobase, although more can also be utilized asdesired. As will be appreciated by those skilled in the art, there maybe a tradeoff between reaction kinetics and length of the linkers; ifthe length of the linker(s) are so long that contact resulting inligation is kinetically disfavored, shorter linkers may be desired.However, in some cases, when kinetics are not important, the length ofthe gap and the resulting linkers may be longer, to allow spanning gapsof 1 to 10 nucleobases. Generally, in this embodiment, what is importantis that the length of the linker(s) roughly corresponds to the number ofnucleobases in the gap.

In another aspect of this embodiment of the invention, the formation ofabasic sites in a ligation product as compared to the target sequenceserves to destabilize the duplex. For example, Abe and Kool (J. Am.Chem. Soc. (2004) 126:13980-13986) compared the turnover when twodifferent 8-mer oligonucleotide probes (Bu42 and DT40) were ligated withthe same 7-mer probe (Thio 4). When Thio4 is ligated with DT40, acontinuous 15-mer oligonucleotide probe with a nearly native DNAstructure is formed that should be perfectly matched with the DNAtarget. However, when Thio4 is ligated with Bu42, a 15-meroligonucleotide probe is formed, but when the probe is bound to thetarget, it has an abasic site in the middle that is spanned by an alkanelinker. Comparison of the melting temperature (Tm) of these two probeswhen bound to the target shows approximately a 12° C. difference inmelting temperature (58.5 for Bu42 versus 70.7° C. for DT40). This 12°C. difference in melting temperature led to roughly a 10-fold increasein product turnover (91.6- Bu42 versus 8.2 DT40) at 25° C. when theprobe sets (10,000-fold excess, 10 μM conc) were present in large excesscompared to the target (1 nM). Similarly, Dose et al (Dose 2006) showedhow a 4° C. decrease in Tm for two identical sequences, chemicallyligated PNA probes (53° C. versus 57° C.) results in approximately a4-fold increase in product turnover.

Recent work has demonstrated the use of chemical ligation based QuenchedAuto-Ligation (QUAL) probes to monitor RNA expression and detect singlebase mismatches inside bacterial and human cells (WO 2004/0101011 hereinincorporated by reference).

In one embodiment, destabilization moieties are based on the removal ofstabilization moieties. That is, if a ligation probe contains a moietythat stabilizes its hybridization to the target, upon ligation andrelease of the stabilization moiety, there is a drop in the stability ofthe ligation product. Accordingly, one general scheme for reducingproduct inhibition is to develop probes that release a molecular entitylike a minor groove binding molecule during the course of the initialchemical ligation reaction or following a secondary reaction postligation. Depending on the oligonucleotide sequence, minor groovebinders like the dihydropyrroloindole tripeptide (DPI₃) described byKutyavin (Kutyavin 1997 and Kutyavin 2000) can increase the Tm of aduplex nucleic acid by up to 40° C. when conjugated to the end of anoligonucleotide probe. In contrast, the unattached version of the DPI₃only increases the Tm of the same duplex by 2° C. or so. Thus, minorgroove binders can be used to produce probe sets with enhanced bindingstrengths, however if the minor groove binder is released during thecourse of the reaction, the binding enhancement is loss and the ligatedproduct will display a decreased Tm relative to probes in which theminor groove binder is still attached.

Suitable minor groove binding molecules include, but are not limited to,dihydropyrroloindole tripeptide (DPI₃), distamycin A, andpyrrole-imidazole polyamides (Gottesfeld, J. M., et al., J. Mol. Biol.(2001) 309:615-629.

In addition to minor groove binding molecules tethered intercalators andrelated molecules can also significantly increase the meltingtemperature of oligonucleotide duplexes, and this stabilization issignificantly less in the untethered state. (Dogan, et al., J. Am. ChemSoc. (2004) 126:4762-4763 and Narayanan, et al., Nucleic Acids Research,(2004) 32:2901-2911).

Similarly, as will be appreciated by those in the art, probes withattached oligonucleotide fragments (DNA, PNA, LNA, etc) capable oftriple helix formation, can serve as stabilization moieties that uponrelease, results in a decrease of stabilization of the ligation productto the target sequence (Pooga, M, et al., Biomolecular Engineering(2001) 17:183-192.

Another general scheme for decreasing product inhibition by lowering thebinding strength of the ligated product is to incorporate abasic sitesat the point of ligation. This approach has been previously demonstratedby Abe (J. Am. Chem. Soc. (2004) 126:13980-13986), however it is alsopossible to design secondary probe rearrangements to further amplify thedecrease in Tm via straining the alignment between the ligated probesand the target. For example, Dose et al. (Org. Letters (2005) 7:204365-4368) showed how a rearrangement post-ligation that changed thespacing between PNA bases from the ideal 12 sigma bonds to 13 resultedin a lowering of the Tm by 4° C. Larger rearrangements and secondaryreactions that interfere with the binding of the product to the targetor result in the loss of oligonucleotide bases can further decrease theTm.

The present invention provides methods and compositions for a ligationreaction that results in a chain contraction of up to 4 sigma bondsduring the rearrangement, which should have a significant effect on theTm post-rearrangement compared to the 1 base expansion using thechemistry described by Dose. This chemistry is based on the acyltransfer auxiliary that has been described previously (Offer et al., JAm Chem Soc. (2002) 124(17):4642-6). Following completion of the chaincontraction, a free-thiol is generated that is capable of undergoinganother reaction either with a separate molecule or with itself. Forexample, this thiol could react with an internal thioester to severelykink the oligonucleotide and thus further decrease the ligationproduct's ability to bind to the target.

Thus, in this embodiment, ligation reactions that release functionalgroups that will undergo a second reaction with the ligation product canreduce stabilization of the hybrid of the ligation product and thetarget sequence.

Additional Functionalities of Ligation Probes

In addition to the target domains, ligation moieties, and optionallinkers, one or more of the ligation probes of the invention can haveadditional functionalities, including, but not limited to, promoter andprimer sequences (or complements thereof, depending on the assay),labels including label probe binding sequences and anchor sequences.Additional functionalities including variable spacer sequences (alsoreferred to as stuffer sequences) are described hereinbelow withreference to the CLPA assay.

In one aspect of the invention, the upstream oligonucleotide probe canhave a promoter site or primer binding site for a subsequent enzymaticamplification reaction. In one embodiment, the upstream probe containsthe promoter sequence for a RNA polymerase, e.g. T7, SP6 or T3. Inanother embodiment, both the upstream and down stream oligonucleotidescontain primer binding sequences. Promoter and primer binding sequencesare designed so as to not interact with the nucleic acid targets to anyappreciable extent. In a preferred embodiment, when detecting multipletargets simultaneously, all of the oligonucleotide probe sets in thereaction are designed to contain identical promoter or primer pairbinding sites such that following ligation and purification, ifappropriate, all of the ligated products can be amplified simultaneouslyusing the same enzyme and/or same primers.

In one embodiment, one or more of the ligation probes comprise apromoter sequence. In embodiments that employ a promoter sequence, thepromoter sequence or its complement will be of sufficient length topermit an appropriate polymerase to interact with it. Detaileddescriptions of sequences that are sufficiently long for polymeraseinteraction can be found in, among other places, Sambrook and Russell.In certain embodiments, amplification methods comprise at least onecycle of amplification, for example, but not limited to, the sequentialprocedures of: interaction of a polymerase with a promoter; synthesizinga strand of nucleotides in a template-dependent manner using apolymerase; and denaturing the newly-formed nucleic acid duplex toseparate the strands.

In another embodiment, one or both of the ligation probes comprise aprimer sequence. As outlined below, the ligation products of the presentinvention may be used in additional reactions such as enzymaticamplification reactions. In one embodiment, the ligation probes includeprimer sequences designed to allow an additional level of amplification.As used herein, the term “primer” refers to nucleotide sequence, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofnucleic acid sequence synthesis when placed under conditions in whichsynthesis of a primer extension product which is complementary to anucleic acid strand is induced, i.e. in the presence of differentnucleotide triphosphates and a polymerase in an appropriate buffer(“buffer” includes pH, ionic strength, cofactors etc.) and at a suitabletemperature. One or more of the nucleotides of the primer can bemodified, for instance by addition of a methyl group, a biotin ordigoxigenin moiety, a fluorescent tag or by using radioactivenucleotides. A primer sequence need not reflect the exact sequence ofthe template. For example, a non-complementary nucleotide fragment maybe attached to the 5′ end of the primer, with the remainder of theprimer sequence being substantially complementary to the target strand.

By using several priming sequences and primers, a first ligation productcan serve as the template for additional ligation products. These primersequences may serve as priming sites for PCR reactions, which can beused to amplify the ligation products. In addition to PCR reactions,other methods of amplification can utilize the priming sequences,including but not limited to ligase chain reactions, InvaderTM,positional amplification by nick translation (NICK), primerextension/nick translation, and other methods known in the art. As usedherein, “amplification” refers to an increase in the number of copies ofa particular nucleic acid. Copies of a particular nucleic acid made invitro in an amplification reaction are called “amplicons” or“amplification products”.

Amplification may also occur through a second ligation reaction, inwhich the primer sites serve as hybridization sites for a new set ofligation probes which may or may not comprise sequences that areidentical to the first set of ligation probes that produced the originalligation products. The target sequence is thus exponentially amplifiedthrough amplification of ligation products in subsequent cycles ofamplification.

In another embodiment of this aspect of the invention, the primersequences are used for nested ligation reactions. In such nestedligation reactions, a first ligation reaction is accomplished usingmethods described herein such that the ligation product can be captured,for example by using biotinylated primers to the desired strand andcapture on beads (particularly magnetic beads) coated with streptavidin.After the ligation products are captured, a second ligation reaction isaccomplished by hybridization of ligation probes to primer sequenceswithin a section of the ligation product which is spatially removed from(i.e., downstream from) the end of the ligation product which isattached to the capture bead, probe, etc. At least one of the primersequences for the secondary ligation reaction will be located within theregion of the ligation product complementary to the ligation probe whichis not the ligation probe that included the anchor or capture sequence.The ligation products from this second ligation reaction will thusnecessarily only result from those sequences successfully formed fromthe first chemical ligation, thus removing any “false positives” fromthe amplification reaction. In another embodiment, the primer sequencesused in the secondary reaction may be primer sites for other types ofamplification reactions, such as PCR.

In one embodiment, one or more of the ligation probes comprise an anchorsequence. By “anchor sequence” herein is meant a component of a ligationprobe that allows the attachment of a ligation product to a support forthe purposes of detection. Suitable means for detection include asupport having attached thereto an appropriate capture moiety.Generally, such an attachment will occur via hybridization of the anchorsequence with a capture probe, which is substantially complementary tothe anchor sequence.

In one embodiment of this aspect of the invention, the upstreamoligonucleotide is designed to have an additional nucleotide segmentthat does not bind to the target of interest, but is to be used tosubsequently capture the ligated product on a suitable solid support ordevice of some sort. In a preferred embodiment of this aspect of theinvention, the downstream oligonucleotide has a detectable labelattached to it, such that following ligation, the resulting product willcontain a capture sequence for a solid support at its 3′ end and adetectable label at its 5′ end, and only ligated products will containboth the capture sequence and the label.

In another aspect of the invention pertaining to multiplex targetdetection, each upstream probe of a probe set may be designed to have aunique sequence at is 3′ end that corresponds to a different position ona DNA array. Each downstream probe of a probe set may optionally containa detectable label that is identical to the other down stream probes,but a unique target binding sequence that corresponds to its respectivetargets. Following hybridization with the DNA array, only ligated probesthat have both an address sequence (upstream probe) and a label(downstream probe) will be observable.

In another aspect of the invention, the detectable label can be attachedto the upstream probe and the capture sequence can be a part of thedownstream probe, such that the ligated products will have thedetectable label more towards the 3′ end and the capture sequencetowards the 5′ end of the ligated product. The exact configuration isbest determined via consideration of the ease of synthesis as well asthe characteristics of the devices to be used to subsequently detect theligated reaction product.

The anchor sequence may have both nucleic and non-nucleic acid portions.Thus, for example, flexible linkers such as alkyl groups, includingpolyethylene glycol linkers, may be used to provide space between thenucleic acid portion of the anchor sequence and the support surface.This may be particularly useful when the ligation products are large.

In addition, in some cases, sets of anchor sequences that correspond tothe capture probes of “universal arrays” can be used. As is known in theart, arrays can be made with synthetic generic sequences as captureprobes, that are designed to non-complementary to the target sequencesof the sample being analyzed but to complementary to the array bindingsequences of the ligation probe sets. These “universal arrays” can beused for multiple types of samples and diagnostics tests because samearray binding sequences of the probes can be reused/paired withdifferent target recognition sequences.

In one embodiment, one or more of the ligation probes comprise a label.By “label” or “labeled” herein is meant that a compound has at least oneelement, isotope or chemical compound attached to enable the detectionof the compound, e.g. renders a ligation probe or ligation or transferproduct detectable using known detection methods, e.g., electronic,spectroscopic, photochemical, or electrochemiluminescent methods.. Ingeneral, labels fall into three classes: a) isotopic labels, which maybe radioactive or heavy isotopes; b) magnetic, electrical, thermal; andc) colored or luminescent dyes; although labels include enzymes andparticles such as magnetic particles as well. The dyes may bechromophores or phosphors but are preferably fluorescent dyes, which dueto their strong signals provide a good signal-to-noise ratio. Suitabledyes for use in the invention include, but are not limited to,fluorescent lanthanide complexes, including those of Europium andTerbium, fluorescein, fluorescein isothiocyanate, carboxyfluorescein(FAM), dichlorotriazinylamine fluorescein, rhodamine,tetramethylrhodamine, umbelliferone, eosin, erythrosin, coumarin,methyl-coumarins, pyrene, Malacite green, Cy3, Cy5, stilbene, LuciferYellow, Cascade Blue™, Texas Red, alexa dyes, dansyl chloride,phycoerythin, green fluorescent protein and its wavelength shiftedvariants, bodipy, and others known in the art such as those described inHaugland, Molecular Probes Handbook, (Eugene, Oreg.) 6th Edition; TheSynthegen catalog (Houston, Tex.), Lakowicz, Principles of FluorescenceSpectroscopy, 2nd Ed., Plenum Press New York (1999), and othersdescribed in the 6th Edition of the Molecular Probes Handbook by RichardP. Haugland, herein expressly incorporated by reference. Additionallabels include nanocrystals or Q-dots as described in U.S. Ser. No.09/315,584, herein expressly incorporated by reference.

In a preferred embodiment, the label is a secondary label that part of abinding partner pair. For example, the label may be a hapten or antigen,which will bind its binding partner. In a preferred embodiment, thebinding partner can be attached to a solid support to allow separationof extended and non-extended primers. For example, suitable bindingpartner pairs include, but are not limited to: antigens (such asproteins (including peptides)) and antibodies (including fragmentsthereof (FAbs, etc.)); proteins and small molecules, includingbiotin/streptavidin; enzymes and substrates or inhibitors; otherprotein—protein interacting pairs; receptor-ligands; and carbohydratesand their binding partners. Nucleic acid—nucleic acid binding proteinpairs are also useful. In general, the smaller of the pair is attachedto the NTP for incorporation into the primer. Preferred binding partnerpairs include, but are not limited to, biotin (or imino-biotin) andstreptavidin, digeoxinin and Abs, and Prolinx™ reagents.

In a preferred embodiment, the binding partner pair comprises biotin orimino-biotin and streptavidin. Imino-biotin is particularly preferred asimino-biotin disassociates from streptavidin in pH 4.0 buffer whilebiotin requires harsh denaturants (e.g. 6 M guanidinium HCl, pH 1.5 or90% formamide at 95° C.).

In a preferred embodiment, the binding partner pair comprises a primarydetection label (for example, attached to a ligation probe) and anantibody that will specifically bind to the primary detection label. By“specifically bind” herein is meant that the partners bind withspecificity sufficient to differentiate between the pair and othercomponents or contaminants of the system. The binding should besufficient to remain bound under the conditions of the assay, includingwash steps to remove non-specific binding. In some embodiments, thedissociation constants of the pair will be less than about 10⁻⁴ to 10⁻⁶M⁻¹, with less than about 10⁻⁵ to 10⁻⁹ M⁻¹ being preferred and 10⁻⁹ M⁻¹being particularly preferred.

In a preferred embodiment, the secondary label is a chemicallymodifiable moiety. In this embodiment, labels comprising reactivefunctional groups are incorporated into the nucleic acid. The functionalgroup can then be subsequently labeled with a primary label. Suitablefunctional groups include, but are not limited to, amino groups, carboxygroups, maleimide groups, oxo groups and thiol groups, with amino groupsand thiol groups being particularly preferred. For example, primarylabels containing amino groups can be attached to secondary labelscomprising amino groups, for example using linkers as are known in theart; for example, homo-or hetero-bifunctional linkers as are well known(see 1994 Pierce Chemical Company catalog, technical section oncross-linkers, pages 155 200, incorporated herein by reference).

In this embodiment, the label may also be a label probe binding sequenceor complement thereof. By “label probe” herein is meant a nucleic acidthat is substantially complementary to the binding sequence and islabeled, generally directly.

Synthetic Methods

The compositions of the invention are generally made using knownsynthetic techniques. In general, methodologies based on standardphosphoramidite chemistries find particular use in one aspect of thepresent invention, although as is appreciated by those skilled in theart, a wide variety of nucleic acid synthetic reactions are known.

Methods of making probes having halo leaving groups is known in the art;see for example Abe et al., Proc Natl Acad Sci USA (2006)103(2):263-8;Silverman et al., Nucleic Acids Res. (2005) 33(15):4978-86; Cuppollettiet al., Bioconjug Chem. (2005) 16(3):528-34; Sando et al., J Am ChemSoc. (2004) 4;126(4):1081-7; Sando et al., Nucleic Acids Res Suppl.(2002) 2:121-2; Sando et al., J Am Chem Soc. (2002) 124(10):2096-7; Xuet al., Nat Biotechnol. (2001) 19(2):148-52; Xu et al., Nucleic AcidsRes. (1998) 26(13):3159-64; Moran et al., Proc Natl Acad Sci USA (1997)94(20):10506-11; Kool, U.S. Pat. No. 7,033,753; Kool, U.S. Pat. No.6,670,193; Kool, U.S. Pat. No. 6,479,650; Kool, U.S. Pat. No. 6,218,108;Kool, U.S. Pat. No. 6,140,480; Kool, U.S. Pat. No. 6,077,668; Kool, U.S.Pat. No. 5,808,036; Kool, U.S. Pat. No. 5,714,320; Kool, U.S. Pat. No.5,683,874; Kool, U.S. Pat. No. 5,674,683; and Kool, U.S. Pat. No.5,514,546, each of which is incorporated herein by reference in itsentirety.

Additional components such as labels, primer sequences, promotersequences, etc. are generally incorporated as is known in the art. Thespacing of the addition of fluorophores and quenchers is well known aswell.

Secondary Reactions

Prior to detecting the ligation or transfer reaction product, there maybe additional amplification reactions. Secondary amplification reactionscan be used to increase the signal for detection of the target sequence;e.g. by increasing the number of ligated products produced per copy oftarget. In one embodiment, any number of standard amplificationreactions can be performed on the ligation product, including, but notlimited to, strand displacement amplification (SDA), nucleic acidsequence based amplification (NASBA), ligation amplification and thepolymerase chain reaction (PCR); including a number of variations ofPCR, including “quantitative competitive PCR” or “QC-PCR”, “arbitrarilyprimed PCR” or “AP-PCR”, “immuno-PCR”, “Alu-PCR”, “PCR single strandconformational polymorphism” or “PCR-SSCP”, “reverse transcriptase PCR”or “RT-PCR”, “biotin capture PCR”, “vectorette PCR”. “panhandle PCR”,and “PCR select cDNA subtraction”, among others. In one embodiment, theamplification technique is not PCR. According to certain embodiments,one may use ligation techniques such as gap-filling ligation, including,without limitation, gap-filling OLA and LCR, bridging oligonucleotideligation, FEN-LCR, and correction ligation. Descriptions of thesetechniques can be found, among other places, in U.S. Pat. No. 5,185,243,published European Patent Applications EP 320308 and EP 439182,published PCT Patent Application WO 90/01069, published PCT PatentApplication WO 02/02823, and U.S. patent application Ser. No.09/898,323.

In addition to standard enzymatic amplification reactions, it ispossible to design probe schemes where the ligated product that isinitially produced can itself be the target of a secondary chemicalligation reaction.

Furthermore, “preamplification reactions” can be done on starting samplenucleic acids to generate more target sequences for the chemicalreaction ligation. For example, whole genome amplification can be done.

Assays

As will be appreciated by those skilled in the art, assays utilizingmethods and compositions of the invention can take on a wide variety ofconfigurations, depending on the desired application, and can include insitu assays (similar to FISH), solution based assays (e.g.transfer/removal of fluorophores and/or quenchers), and heterogeneousassays (e.g. utilizing solid supports for manipulation, removal and/ordetection, such as the use of high density arrays). In addition, assayscan include additional reactions, such as pre-amplification of targetsequences and secondary amplification reactions after ligation hasoccurred, as is outlined herein.

Assays pertaining to this aspect of the invention, as described herein,may rely on increases in a signal, e.g. the generation of fluorescenceor chemiluminescence. However, as will be appreciated by those in theart, assays that rely on decreases in such signals are also possible.

In one embodiment, assay reactions are performed “in situ” (alsoreferred to in various assay formats as “in vitro” and/or “ex vivo”depending on the sample), similar to FISH reactions. Since no exogeneousenzymes need be added, reagents can be added to cells (living,electroporated, fixed, etc.) such as histological samples for thedetermination of the presence of target sequences, particularly thoseassociated with disease states or other pathologies.

In addition, “in vitro” assays can be done where target sequences areextracted from samples. Samples can be processed (e.g. for paraffinembedded samples, the sample can be prepared), the reagents added andthe reaction allowed to proceed, with detection following as is done inthe art.

In one embodiment, ligated products are detected using solid supports.For example, the ligated products are attached to beads, using eitheranchor probe/capture probe hybridization or other binding techniques,such as the use of a binding partner pair (e.g. biotin andstreptavidin). In one embodiment, a transfer reaction results in abiotin moiety being transferred from the first ligation probe to asecond ligation probe comprising a label. Beads comprising streptavidinare contacted with the sample, and the beads are examined for thepresence of the label, for example using FACS technologies.

In other embodiments, ligated products are detected using heterogeneousassays. That is, the reaction is done in solution and the product isadded to a solid support, such as an array or beads. Generally, oneligation probe comprises an anchor sequence or a binding pair partner(e.g. biotin, haptens, etc.) and the other comprises a label (e.g. afluorophore, a label probe binding sequence, etc.). The ligated productis added to the solid support, and the support optionally washed. Inthis embodiment, only the ligated product will be captured and belabeled.

In another aspect of the invention, one of oligonucleotide probes has anattached magnetic bead or some other label (biotin) that allows for easymanipulation of the ligated product. The magnetic bead or label can beattached to either the upstream or the downstream probe using any numberof configurations as outlined herein.

As described herein, secondary reactions can also be done, whereadditional functional moieties (e.g. anchor sequences, primers, labels,etc.) are added. Similarly, secondary amplification reactions can bedone as described herein.

Detection systems are known in the art, and include optical assays(including fluorescence and chemiluminescent assays), enzymatic assays,radiolabelling, surface plasmon resonance, magnetoresistance, cantileverdeflection, surface plasmon resonance, etc. In some embodiments, theligated product can be used in additional assay technologies, forexample, as described in 2006/0068378, hereby incorporated by reference,the ligated product can serve as a linker between light scatteringparticles such as colloids, resulting in a color change in the presenceof the ligated product.

In some embodiments, the detection system can be included within thesample collection tube; for example, blood collection devices can haveassays incorporated into the tubes or device to allow detection ofpathogens or diseases.

Solid Supports

As outlined above, the assays can be run in a variety of ways. In assaysthat utilize detection on solid supports, there are a variety of solidsupports, including arrays, that find use in the invention.

In some embodiments, solid supports such as beads find use in thepresent invention. For example, binding partner pairs (one on theligated product and one on the bead) can be used as outlined above toremove non-ligated reactants. In this embodiment, magnetic beads areparticularly preferred.

In some embodiments of the invention, capture probes are attached tosolid supports for detection. For example, capture probes can beattached to beads for subsequent analysis using any suitable technique,e.g. FACS. Similarly, bead arrays as described below may be used.

In one embodiment, the present invention provides arrays, each arraylocation comprising at a minimum a covalently attached nucleic acidprobe, generally referred to as a “capture probe”. By “array” herein ismeant a plurality of nucleic acid probes in an array format; the size ofthe array will depend on the composition and end use of the array.Arrays containing from about 2 different capture ligands to manythousands can be made. Generally, for electrode-based assays, the arraywill comprise from two to as many as 100,000 or more, depending on thesize of the electrodes, as well as the end use of the array. Preferredranges are from about 2 to about 10,000, with from about 5 to about 1000being preferred, and from about 10 to about 100 being particularlypreferred. In some embodiments, the compositions of the invention maynot be in array format; that is, for some embodiments, compositionscomprising a single capture probe may be made as well. In addition, insome arrays, multiple substrates may be used, either of different oridentical compositions. Thus, for example, large arrays may comprise aplurality of smaller substrates. Nucleic acid arrays are known in theart, and can be classified in a number of ways; both ordered arrays(e.g. the ability to resolve chemistries at discrete sites), and randomarrays (e.g. bead arrays) are included. Ordered arrays include, but arenot limited to, those made using photolithography techniques (e.g.Affymetrix GeneChip®), spotting techniques (Synteni and others),printing techniques (Hewlett Packard and Rosetta), electrode arrays,three dimensional “gel pad” arrays and liquid arrays.

In a preferred embodiment, the arrays are present on a substrate. By“substrate” or “solid support” or other grammatical equivalents hereinis meant any material that can be modified to contain discreteindividual sites appropriate for the attachment or association ofnucleic acids. The substrate can comprise a wide variety of materials,as will be appreciated by those skilled in the art, including, but notlimited to glass, plastics, polymers, metals, metalloids, ceramics, andorganics. When the solid support is a bead, a wide variety of substratesare possible, including but not limited to magnetic materials, glass,silicon, dextrans, and plastics.

Hardware

Microfluidics

In another aspect of the invention, a fluidic device similar to thosedescribed by Liu (2006) is used to automate the methodology described inthis invention. See for example U.S. Pat. No. 6,942,771, hereinincorporated by reference for components including but not limited tocartridges, devices, pumps, wells, reaction chambers, and detectionchambers. The fluidic device may also include zones for capture ofmagnetic particles, separation filters and resins, including membranesfor cell separation (i.e.Leukotrap™ from Pall). The device may includedetection chambers for in-cartridge imaging of fluorescence signalgenerated during Real-Time PCR amplification (i.e. SYBR green, Taqman,Molecular Beacons), as well as capillary electrophoresis channels foron-device separation and detection of reactions products (amplicons andligation products). In a preferred embodiment, the capillaryelectrophoresis channel can be molded in a plastic substrate and filledwith a sieving polymer matrix (POP-7™ from Applied Biosystems). Channelscontaining non-sieving matrix can also be used with properly designedprobe sets.

In a preferred embodiment, the devices of the invention comprise liquidhandling components, including components for loading and unloadingfluids at each station or sets of stations. The liquid handling systemscan include robotic systems comprising any number of components. Inaddition, any or all of the steps outlined herein may be automated;thus, for example, the systems may be completely or partially automated.

As will be appreciated by those in the art, there are a wide variety ofcomponents which can be used, including, but not limited to, one or morerobotic arms; plate handlers for the positioning of microplates; holderswith cartridges and/or caps; automated lid or cap handlers to remove andreplace lids for wells on non-cross contamination plates; tip assembliesfor sample distribution with disposable tips; washable tip assembliesfor sample distribution; 96 well loading blocks; cooled reagent racks;microtitler plate pipette positions (optionally cooled); stacking towersfor plates and tips; and computer systems.

Fully robotic or microfluidic systems include automated liquid-,particle-, cell- and organism-handling including high throughputpipetting to perform all steps of screening applications. This includesliquid, particle, cell, and organism manipulations such as aspiration,dispensing, mixing, diluting, washing, accurate volumetric transfers;retrieving, and discarding of pipet tips; and repetitive pipetting ofidentical volumes for multiple deliveries from a single sampleaspiration. These manipulations are cross-contamination-free liquid,particle, cell, and organism transfers. This instrument performsautomated replication of microplate samples to filters, membranes,and/or daughter plates, high-density transfers, full-plate serialdilutions, and high capacity operation.

In a preferred embodiment, chemically derivatized particles, plates,cartridges, tubes, magnetic particles, or other solid phase matrix withspecificity to the assay components are used. The binding surfaces ofmicroplates, tubes or any solid phase matrices include non-polarsurfaces, highly polar surfaces, modified dextran coating to promotecovalent binding, antibody coating, affinity media to bind fusionproteins or peptides, surface-fixed proteins such as recombinant proteinA or G, nucleotide resins or coatings, and other affinity matrix areuseful in this invention.

In a preferred embodiment, platforms for multi-well plates, multi-tubes,holders, cartridges, minitubes, deep-well plates, microfuge tubes,cryovials, square well plates, filters, chips, optic fibers, beads, andother solid-phase matrices or platform with various volumes areaccommodated on an upgradable modular platform for additional capacity.This modular platform includes a variable speed orbital shaker, andmulti-position work decks for source samples, sample and reagentdilution, assay plates, sample and reagent reservoirs, pipette tips, andan active wash station.

In a preferred embodiment, thermocycler and thermoregulating systems areused for stabilizing the temperature of heat exchangers such ascontrolled blocks or platforms to provide accurate temperature controlof incubating samples from 0 .degree. C. to 100 .degree. C.; this is inaddition to or in place of the station thermocontrollers.

In a preferred embodiment, interchangeable pipet heads (single ormulti-channel) with single or multiple magnetic probes, affinity probes,or pipetters robotically manipulate the liquid, particles, cells, andorganisms. Multi-well or multi-tube magnetic separators or platformsmanipulate liquid, particles, cells, and organisms in single or multiplesample formats.

In some embodiments, the instrumentation will include a detector, whichcan be a wide variety of different detectors, depending on the labelsand assay. In a preferred embodiment, useful detectors include amicroscope(s) with multiple channels of fluorescence; plate readers toprovide fluorescent, electrochemical and/or electrical impedanceanalyzers, ultraviolet and visible spectrophotometric detection withsingle and dual wavelength endpoint and kinetics capability,fluroescence resonance energy transfer (FRET), luminescence, quenching,two-photon excitation, and intensity redistribution; CCD cameras tocapture and transform data and images into quantifiable formats;capillary electrophoresis systems, mass spectrometers and a computerworkstation.

These instruments can fit in a sterile laminar flow or fume hood, or areenclosed, self-contained systems, for cell culture growth andtransformation in multi-well plates or tubes and for hazardousoperations. The living cells may be grown under controlled growthconditions, with controls for temperature, humidity, and gas for timeseries of the live cell assays. Automated transformation of cells andautomated colony pickers may facilitate rapid screening of desiredcells.

Flow cytometry or capillary electrophoresis formats can be used forindividual capture of magnetic and other beads, particles, cells, andorganisms.

The flexible hardware and software allow instrument adaptability formultiple applications. The software program modules allow creation,modification, and running of methods. The system diagnostic modulesallow instrument alignment, correct connections, and motor operations.The customized tools, labware, and liquid, particle, cell and organismtransfer patterns allow different applications to be performed. Thedatabase allows method and parameter storage. Robotic and computerinterfaces allow communication between instruments.

In a preferred embodiment, the robotic apparatus includes a centralprocessing unit which communicates with a memory and a set ofinput/output devices (e.g., keyboard, mouse, monitor, printer, etc.)through a bus. Again, as outlined below, this may be in addition to orin place of the CPU for the multiplexing devices of the invention. Thegeneral interaction between a central processing unit, a memory,input/output devices, and a bus is known in the art. Thus, a variety ofdifferent procedures, depending on the experiments to be run, are storedin the CPU memory.

These robotic fluid handling systems can utilize any number of differentreagents, including buffers, reagents, samples, washes, assay componentssuch as label probes, etc.

Kits

In another aspect of the invention, a kit for the routine detection of apredetermined set of nucleic acid targets is produced that utilizesprobes, techniques, methods, and a chemical ligation reaction asdescribed herein as part of the detection process. The kit can compriseprobes, target sequences, instructions, buffers, and/or other assaycomponents.

Chemical Ligation Dependent Probe Amplification (CLPA)

In another embodiment, the invention relates to chemical ligationdependent probe amplification (CLPA) technology. CLPA is based on thechemical ligation of target specific oligonucleotide probes to form aligation product. This ligation product subsequently serves as atemplate for an enzymatic amplification reaction to produce ampliconswhich are subsequently analyzed using any suitable means. CLPA can beused for a variety of purposes including but not limited to analysis ofcomplex gene signature patterns. Unlike other techniques such as DASL(Bibikova, M., et al., American Journal of Pathology, (2004), 165:5,1799-1807) and MLPA (Schouten, U.S. Pat. No. 6,955,901) which utilize anenzymatic ligation reaction, CLPA uses a chemical ligation reaction.

In one embodiment, the CLPA assay comprises the use of oligonucleotideprobe pairs that incorporate reactive moieties that can self-ligate whenproperly positioned on a target sequence. In a preferred embodiment, a3′-phosphorothioate moiety on one probe reacts with a 5′-DABSYL leavinggroup on the other probe (See Scheme 1 and FIG. 6).

The 5′-DABSYL group reacts about four times faster than other moieties,e.g. iodine, and also simplifies purification of the probes duringsynthesis.

CLPA has several distinct advantages over other sequence-basedhybridization techniques. First, CLPA can be applied directly to RNAanalysis without the need to make a DNA copy beforehand. Second, CLPA isrelatively insensitive to sample contaminants and can be applieddirectly to impure samples including body samples such as blood, urine,saliva and feces. Third, CLPA involves fewer steps than other knownmethods, thereby reducing the time required to gain a result. Moreover,CLPA probes can be stored dry, and properly designed systems willspontaneously react to join two or more oligonucleotides in the presenceof a complementary target sequence. Chemical ligation reactions showexcellent sequence selectivity and can be used to discriminate singlenucleotide polymorphisms.

Significantly, unlike enzymatic ligation methods, CLPA shows nearlyidentical reactivity on DNA and RNA targets which, as described morefully below, renders CLPA more efficient that other known systems, andexpands the scope of applications to which CLPA can be utilized.

Advantageously, the CLPA assay reduces the number of steps required toachieve a result, which provides the potential to achieve results insignificantly shorter time periods. For example, the general processflow for a standard reverse transcriptase (RT)- multiplexligase-dependent probe ligation (MLPA) involves the following steps:

1. Isolate total RNA.

2. Use Reverse Transcriptase to make cDNA copy.

3. Hybridize MLPA probe sets to the cDNA target overnight.

4. Add DNA Ligase to join target-bound probes.

5. Amplify ligated probes, e.g. PCR amplification using Taq polymeraseand fluorescently labeled PCR primers.

6. Analyze the sample, for example, by CE.

Unlike standard RT-MLPA, CLPA enables analysis to be carried outdirectly on cell and blood lysates and on RNA targets. Thus, unlike aRT-MLPA, CLPA avoids the necessity of having to isolate the RNA, andthen perform reverse transcription to make a cDNA copy prior toligation. This shortens the time for achieving a result and provides ameans to achieve faster analysis.

A further advantage of CLPA is that incorporation of a capture moiety onone probe enables a rapid and specific method for purification of theresulting ligation product from the crude sample free of all impuritiesand non-target nucleic acid materials, as described below for abiotin-labeled probe. This capability is particularly advantageous inapplications where the target nucleic acid is found in the presence of alarge excess of non-target nucleic acid, such as in detection ofinfectious agents (bacteria, fungi, viruses). In this case, the presenceof large amounts of host nucleic acid requires use of a high-capacityextraction method, which in turn can result in inefficient amplificationof the target nucleic acid due to large amounts of non-target nucleicacid and/or carry-over of inhibitory contaminants.

In another embodiment of this aspect of the invention, faster reactiontimes are further facilitated by driving the hybridization reaction withhigher probe concentrations. Thus, for example, input probe sets may beincorporated in the CLPA reaction at relatively high concentrations, forexample, approximately 100-fold higher than those typically used in anMLPA reaction. Elevating the probe concentration significantly reducesthe time required for the hybridization step, typically from overnightto between about 15 minutes to about 1 hour.

When higher probe concentrations are used it is generally preferred toincorporate a purification step prior to amplification, especially forhigh multiplex analysis (e.g. greater than about 5 targets). In oneembodiment of this aspect of this invention, a solid support basedcapture methodology can be employed including membrane capture, magneticbead capture and/or particle capture. In a preferred embodiment, abiotin/streptavidin magnetic bead purification protocol is employedafter ligation and prior to enzymatic amplification. In some instances,the magnetic particles can be directly added to the amplification mastermix without interfering with the subsequent amplification reaction. Inother instances, it is preferable to release the capturedoligonucleotide from the beads and the released oligonucleotide solutionis subsequently amplified without the capture particle or surface beingpresent.

In a preferred embodiment, CLPA involves hybridization of a set ofprobes to a nucleic acid target sequences such that the probes canundergo self-ligation without addition of a ligase. After a ligationproduct is produced, amplification is generally preferred to facilitatedetection and analysis of the product. For this purpose, probes arepreferably designed to incorporate PCR primers such as, e.g. universalPCR primers. In a preferred embodiment, the universal primers are notadded until after the ligation portion of the reaction is complete, andthe primers are added after surface capture purification along with thepolymerase, often as part of a PCR master mix.

The CLPA probes possess reactive moieties positioned such that when theCLPA probes are bound to the nucleic acid target, the reactive moietiesare in close spatial orientation and able to undergo a ligation reactionwithout the addition of enzyme. In a preferred embodiment, the chemicalligation moieties are chosen so as to yield a ligated reaction productthat can be efficiently amplified by the amplification enzyme which isoften a DNA polymerase. Without being bound by theory, chemical ligationchemistries and probe set designs that produce reaction products thatmore closely resemble substrates that are known as being able to beamplified by DNA and RNA polymerases are more likely to yield efficientprobe sets that can be used in the CLPA assay. Especially preferredreaction chemistries are chemical moieties that yield reaction productsthat closely resemble native DNA such as illustrated in Scheme 1involving a reaction between a 3′-phosphorothioate and a 5′ DABSYLleaving group. In another preferred embodiment, probes sets comprise a3′-diphosphorothioate (Miller, G. P. et al, Bioorganic and MedicinalChemistry, (2008) 16:56-64) and a 5′-DABSYL leaving group.

The CLPA probes also incorporate a stuffer sequence (also referred toherein as a variable spacer sequence) to adjust the length of theligation product. As described further below, length variation providesa convenient means to facilitate analysis of ligation product(s). Thestuffer can be located on either probe, though for convenience it isgenerally incorporated on the S probe (3′-phosphorothioate probe).

In one embodiment of this aspect of the invention, CLPA-CE, the stuffersequence is varied in length in order to produce one or more variablelength ligation products which provide the basis for detection andidentification of specific target sequences based on length variation.In a preferred embodiment, variable length ligation products areanalyzed by capillary electrophoresis (CE). Generally stuffer sequencesare included such that the length of different ligation products variesin a range of at least 1 base pair to about 10 base pairs; preferablyfrom 1 base pair to 4 base pairs. In a preferred embodiment, the lengthof the different ligation products vary from approximately 80 bp toabout 400 bp; preferably in a range of about 100 bp to about 300 bp;more preferably in a range of about 100 bp to about 200 bp

In another embodiment, CLPA probes may also contain other optionalelement(s) to facilitate analysis and detection of a ligated product.For example, it is preferred that one of the probes for use in anembodiment herein referred to as CLPA-MDM incorporate an array bindingsequence to bind to an appropriate capture sequence on a microarrayplatform. For CLPA-MDM, the different CLPA reaction products are notseparated by size differences but by the differences in the arraybinding sequence. In this embodiment, the sequence of the array bindingsequence is varied so that each CLPA probe will bind to a unique site ona DNA microarray. The length of the array binding sequence in CLPA-MDMusually varies from 15 to 150 bases, more specifically from 20 to 80bases, and most specifically from 25 to 50 bases. In some embodiments,CLPA probes preferably also include other elements to facilitatepurification and/or analysis including but not limited to labels such asfluorescent labels and hapten moieties such as, for example, biotin forpurifying or detecting ligation product(s). For example, probes and/orligation product(s) that incorporate biotin can be purified on anysuitable avidin/streptavidin platform including beads. Whilebiotin/avidin capture systems are preferred, other hapten systems (e.g.Digoxigenin (DIG) labeling) can be used, as canhybridization/oligonucleotide capture. Hybridization/oligonucleotidecapture is a preferred method when it it desirable to release thecapture product from the beads at a later stage. In addition to magneticbeads, anti-hapten labeled supports (filter paper, porous filters,surface capture) can be used.

CLPA probe-labeling can be on either probe, either at the end orinternally. Preferably biotin is incorporated at the 5′ end on thephosphorothioate (S-probe).

CLPA probes are generally incorporated in a reaction at a concentrationof 250 nanomolar (nM) to 0.01 pM (picomolar) for each probe. Generally,the concentration is between about 1 nM to about 1 pM. Factors toconsider when choosing probe concentration include the particular assayand the target being analyzed. The S-or phosphorothioate or Nucleophileprobe and L- or leaving group or DABSYL containing probes areincorporated at a concentration that equals or exceeds the concentrationof the target. Total concentration of S- and L-probes can reach as highas 10 micromolar (uM). As a non-limiting example, 1 nM for each S and Lprobe×250 CLPA probe pairs would equal 500 nm (1 nm per probe×2 probesper pair×250 targets) at 10 nM for each probe would mean a totalconcentration of 5 uM.

The target concentration usually ranges from about 10 micrograms oftotal RNA to about 10 nanograms, but it can be a little as a single copyof a gene.

In a preferred embodiment of CLPA technology, a CLPA probe set consistsof 2 oligonucleotide probes with complementary reactive groups (FIGS. 1and 2). In another embodiment, the CLPA probe set may consist of 3 ormore probes that bind adjacent to each other on a target. In a preferredembodiment of the 3-probe CLPA reaction, the outer probes are designedto contain the enzymatic amplification primer binding sites, and theinner probe is designed to span the region of the target between theother probes. In a more preferred embodiment, the outer probes havenon-complementary reactive groups such that they are unable to reactwith each other in the absence of the internal (middle) probe (FIG. 3).In some instances, both outer probes may have similar reactive moietiesexcept that one group is at the 5′ end of one probe and the 3′-end ofthe other probe, and the L-probe chemistries may also be similar to eachother except for positioning on the probe. As is known to one who isskilled in the art, different chemical reagents and processes may beneeded to manufacture the probes for the 3-probe CLPA reaction comparedto the probes for the 2-probe CLPA system.

In a preferred embodiment of the 3-probe CLPA system, one outer probecontains a 3′ phosphorothioate (3′ S-probe), the other outer probecontains a 5′-phosphorothioate (5′-S-probe) and the center probecontains both a 3′- and a 5′-DABSYL leaving group. The manufacture of a5′-DABSYL leaving group probes has been reported previously (Sando etal, J. Am. Chem. Soc., (2002), 124(10) 2096-2097). We recently developeda new DNA synthesis reagent that allows for the routine incorporation ofa 3′-DABSYL leaving group (FIG. 4).

CLPA-CE

In one embodiment, CLPA ligation product(s) are detected by sizedifferentiation capillary electrophoresis (CE) on a sieving matrix, orby slab gel electophoresis. A schematic representation for CLPA-CE isprovided in FIG. 1. In this example, analysis is performed directly on ablood sample following cell lysis by any appropriate means includingchemically, mechanically or osmotically, and addition of appropriatelydesigned probes. In a preferred embodiment, chemical lysis of the cellsis used. FIG. 6 provides a general schematic representation of thedesign of a probe set for CLPA-CE analysis. In this example the S probeis designed to include a universal PCR primer for subsequentamplification of ligation product(s); a stuffer sequence which isdesigned with a length that correlates with a specific target; and atarget binding sequence. Likewise, the L-probe includes a target bindingsequence and universal primer. The probes are usually labeled with aflurophore (FAM, Cy3, Cy5, etc), however they can also be detectedwithout fluorescence labeling. The labeling is done by using afluorescently labeled PCR primer.

In this example of CLPA-CE probes, the S probe also includes a biotinmoiety at the 5′ end to facilitate purification and removal of unligatedprobe. Following amplification of ligated product(s), each having aunique length, the reaction mixture is separated by CE, or othersuitable size separation technique. The peak height or intensity of eachproduct is a reflection of target sequence expression, i.e. level oftarget in the sample. (FIG. 1 and FIG. 7).

CLPA-MDM

In another embodiment of this aspect of the invention, CLPA ligationproducts are analyzed/detected by microarray analysis (CLPA-MDM). Aschematic representation of CLPA-MDM is provided in FIG. 2. CLPA-MDMdiffers from CLPA-CE in at least the following respects. First, theprobe sets differ in design. For example, a general representation of aCLPA- MDM probe set is depicted in FIG. 2. As with CLPA-CE probes,CLPA-MDM probe sets can include universal primers for amplification ofligation product(s). They also include target specific sequences, aswell as ligation moieties for enzyme-independent ligation. Additionally,CLPA-MDM probes also may include a stuffer sequence, however the purposeof this stuffer sequence is to adjust the size of the CLPA-MDM to thesame length in an effort to standardize enzymatic amplificationefficiency. Normalization of amplicon size is not a requirement but apreferred embodiment. A second difference between the design of CLPA-CEand CLPA-MDM probe sets is that the latter include a unique arraybinding sequence for use with an appropriate microarray platform.

In respect of the CLPA-MDM aspect of the invention, a microarray bindingsite (ABS sequence) is incorporated into the probe designs for use witha “universal” microarray platform for the detection. Similar to theCLPA-CE system, probes are preferably labeled with a fluorophore, forexample by using a fluorescently labeled PCR primer. Alternatively, forexample, a sandwich assay labeling technique can be used for the finalread-out. Sandwich assays involve designing the probes with a common(generic) label binding site (LBS) in place or in addition to thestuffer sequence and using a secondary probe that will bind to this siteduring the array hybridization step. This methodology is particularlyuseful when it is desirable to label the arrays with a chemiluminescentsystem like a horse radish peroxidase (HRP) labeled oligonucleotide, orwith an electrochemical detection system. Generally, planar microarraysare employed (e.g. microarrays spotted on glass slides or circuit board)for the read-out. However, bead microarrays such as those available fromLuminex and Illumina can also be used (e.g. Luminex xMAP/xtag).

EXAMPLE 1 Quantitative Multiplex Detection of 5 Targets

Multiplex CLPA reactions were performed using five (5) DNA target mimics(corresponding to portions of the MOAP1 (SEQ ID NO:5), PCNA (SEQ IDNO:9), DDB2 (SEQ ID NO:12), BBC3 (SEQ ID NO:16) and BAX (SEQ ID NO:19)genes) combined in one reaction in the presence of their respective CLPAprobes (Table 1) (S and L probes at 1 nM each). The target mimics werepooled in different concentration as shown in Table 2. The targetmimics, S probes and L probes were incubated in PCR buffer (1× PCRbuffer is 1.5 mM MgCL2, 50 mM KCl, 10 mM Tris-HCl pH8.3) for 1 hour at50 C. A 1 ul aliquot of each reaction mixture was used as template forPCR amplification using Dynamo SYBR green PCR mix in the presence ofUniversal Primers (SEQ ID NOS 1 and 2, 300 nM). The samples were PCRcycled for 27 cycles (95 C 15 min followed by 27 cycles of 95 C (10 s),60 C (24 s), 72 C (10 s). After PCR amplification, the samples weredenatured and injected into an ABI 3130 DNA sequencer (capillaryelectrophoresis instrument). The CE trace from the ABI for the 3 samplesas well as a plot of the peak versus target mimic concentration of PCNAis shown in FIG. 7 and a plot of the linear response of the signal ofPCNA as a function on input concentration is shown in FIG. 8.

TABLE 1 Probe and target sequence information. SEQ Amplicon ID NameSequence Detail Size  1 Forward PCR Primer FAM-GGGTTCCCTAAGGGTTGGA  2Reverse PCR Primer GTGCCAGCAAGATCCAATCTAGA  3 MOAP1-LLTACATCCTTCCTAGTCAATTACACTCTAGATTGGA 47 TCTTGCTGGCAC  4 MOAP1-S5′-Biotin- 41 GGGTTCCCTAAGGGTTGGATAGGTAAAT GGCAGTGTAGAACSLigated MOAP1 Amplicon 88  5 MOAP1-TargetGTGTAATTGACTAGGAAGGATGTAGTTCTACACTG mimic CCATTTACCTA  6MOAP1-RNA Target GUGUAAUUGACUAGGAAGGAUGUAGUUCUACAC mimic UGCCAUUUACCUA 7 PCNA-L LTGGTTTGGTGCTTCAAATACTCTCTAGATTGGATC 45 TTGCTGGCAC  8 PCNA-SBiotin- 63 GGGTTCCCTAAGGGTTGGATCGAGTCTACAGATCCCCAACTTTCATAGTCTGAAACTTTCTCCS Ligated PCNA Amplicon 108  9PCNA-Target Mimic AGTATTTGAAGCACCAAACCAGGAGAAAGTTTCA GACTATGA 10 DDB2-LLTAGCAGACACATCCAGGCTCTAGATTGGATCTTG 51 CTGGCAC 11 DDB2-S Biotin- 49GGGTTCCCTAAGGGTTGGATCGAGTCTACTCCAAC TTTGACCACCATTCGGCTACSLigated DDB2 Amplicon 96 12 DDB2-Target MimicGCCTGGATGTGTCTGCTAGTAGCCGAATGGTGGTC A 13 DDB2-RNA TargetGCCUGGAUGUGUCUGCUAGUAGCCGAAUGGUGG Mimic UCA 14 BBC3-LLTCCGAGATTTCCCCCTCTAGATTGGATCTTGCTGG 38 CAC 15 BBC3-S Biotin- 37GGGTTCCCTAAGGGTTGGATCCCAGACTCCTCCCT CTS Ligated BBC3 Amplicon 75 16BBC3-Target Mimic GGG GGA AAT CTC GGA AGA GGG AGG AGT CTG GG 17 BAX-LLTCACGGTCTGCCACGCTCTAGATTGGATCTTGCTG 39 GCAC 18 BAX-SBiotin-GGGTTCCCTAAGGGTTGGA TGA GTC TAC 53 ATGA TC CT TCCCGCCACAAAGATGGSLigated BAX Amplicon 92 19 BAX-Target MimicCGTGGCAGACCGTGACCATCTTTGTGGCGGGA 20 3-phosphorothioate Biotin- 51 GAPDHGGGTTCCCTAAGGGTTGGACGGACGCCTGCTTCAC CACCTTCTTGATGTCAS 21 Middle 2L probeLTCATATTTGGCAGGTTTTTCTAGACGGCAGGTL 32 GAPDH 22 5′-phosphorothioateSCAGGTCCACCACTGACACGTTGGCAGTTCTAGAT 50 GAPDH TGGATCTTGCTGGCACLigated 3-probe amplicon 133 24 GAPDH TargetACT GCC AAC GTG TCA GTG GTG GAC CTG ACC MimicTGC CGT CTA GAA AAA CCT GCC AAA TAT GATGAC ATC AAG AAG GTG GTG AAG CAG GCG TC 25 GAPDH 3-LLTTTTCTAGACGGCAGGTCAGGTCCACCAGATGAT CGACGAGACACTCTCGCCATCTAGATTGGATCTTGCTGGCAC 26 GAPDH 3-S GGGTTCCCTAAGGGTTGGACGGACCAACTCCTCGCCATATCATCTGTACACCTTCTTGATGTCATCATATT TGGCAGGTS 27 GAPDH-3-FAM/BHQ-1(FAM)ccaactcctcgccatatcatctgtacaccttc Taqman Probe ttg(BHQ-1) 28GAPDH 4-L LTGCTGATGATCTTGAGGCTGTTGTCATACTGATGATCGACGAGACACTCTCGCCATCTAGATTGGATCT TGCTGGCAC 29 GAPDH-4-SGGGTTCCCTAAGGGTTGGACGATGGAGTTGATGCT GACGGAAGTCATAGTAAGCAGTTGGTGGTGCAGGAGGCATS 30 GAPDH-4-QUASAR (Quasar 670)tgctgacggaagtcatagtaagca670/BHQ-2 Taqman gttggt(BHQ-2) Probe 31 PCNA 2-LLTCCTTGAGTGCCTCCAACACCTTCTTGAGGATGAT CGACGAGACACTCTCGCCATCTAGATTGGATCTTGCTGGCAC 32 PCNA 2-S GGGTTCCCTAAGGGTTGGACGGTACAACAAGACCCAGCTGACGACTCTTAATATCCCAGCAGGCCTCGTT GATGAGGS 33 PCNA 2-Cal Fluor(CAL Red 610)ctgacgactcttaatatcccagc Orange 560/BHQ-1 aggcctcgtt(BHQ-2)34 DDB2-2-L LTTAGTTCCAAGATAACCTTGGTTCCAGGCTGATGATCGACGAGACACTCTCGCCATCTAGATTGGATCT TGCTGGCAC 35 DDB2-2-SBiotinGGGTTCCCTAAGGGTTGGACGTTAGACGCCAATAGGAGTTTCACTGGTGGCTACCACCCACTGAGA GGAGAAAAGTCATS 36 DDB2-2-(CAL Fluor(Cal Orange 560)cgccaataggagtttcact Orange 560/BHQ-1 ggtggctacca(BHQ-2)L = DABSYL ligation moiety S = phosphorothiate moiety

TABLE 2 Sample Concentrations Sample Target Mimic Concentrations 1 AllTarget mimics at 10 pM final Concentration 2 MOAP1, DDB2 and BBC3 at 10pM, PCNA at 5 pM and BAX at 2 pM 3 MOAP1, DDB2 and BBC3 at 10 pM, PCNAat 1 pM and BAX at 0.5 pM

EXAMPLE 2 CLPA Reactions Using MOAP1 and DDB2 DNA and RNA Target Mimics

Reactions were prepared in duplicate as presented in Table 3 using DNAor RNA target mimics for the MOAP1 and DDB2 genes and CLPA probes setsdesigned to target the sequences. The probe numbers refer to the SEQ IDNOs in Table 1. The reagents were added in the concentrations andvolumes shown in Table 4. The respective S-probe, L-probe and targetmimic were heated to 50° C. for 60 minutes in a 0.2 mL PCR tube, afterwhich 2.5 μl of the CLPA reaction was used as template in a real-timePCR reaction with 40 amplification cycles. Real-time PCR data wasaveraged for the duplicate samples and is presented in Table 3 (Ct valuecolumn). Minimal differences in Ct value between RNA and DNA targetmimics were observed indicating similar probe ligation efficiency on RNAand DNA substrates.

TABLE 3 CLPA Probe Sets. L-Probe S-Probe Target Mimic (1 nM) (1 nM) (10pM) Ct Sample Identifier SEQ ID NO SEQ ID NO SEQ ID NO value 1 MOAP-1 34 5 19.5 DNA 2 MOAP-1 3 4 6 20 RNA 3 DDB2 10 11 12 21 DNA 4 DDB2 10 1113 21 RNA

TABLE 4 Reagent table-Example 1 1X PCR Buffer Buffer* 12.5 ul S-Probe (1nM) & L-Probe (1 nM) 2.5 ul each Target Mimic (100 pM) 2.5 ul Water 5.0ul Heat at 50 C. for 1 hour *1X PCR buffer is 1.5 mM MgCL2, 50 mM KCl,10 mM Tris-HCl pH 8.3

EXAMPLE 3 Direct Analysis of DDB2 RNA Transcripts in Lysis Buffer andLysed Blood

DDB2 messenger RNA (mRNA) was prepared using a in-vitro transcriptionkit from Ambion and a cDNA vector plasmid from Origene (SC122547). Theconcentration of mRNA was determined using PicoGreen RNA assay kit frominvitrogen. The DDB2 probe sets (Table 5) were tested with differentconcentrations of DDB2 mRNA transcript spiked into either water or wholeblood. The reactions mixture components are listed in Table 5. Samples1-4 consisted of DDB2 transcript at 10 ng, 1 ng, 0.1 ng and 0.01 ng inwater, and samples 5-8 consisted of the same concentration range spikedinto whole blood. Similar reactions protocols were followed with theexception of adding Proteinase K to the blood samples so as to reduceprotein coagulation. The procedure is as follows: The reagents wereadded in the concentrations and volumes in Table 5. The S-probes, mRNAtranscript, Guanidine hydrochloride lysis buffer and either water(samples 1-4) or whole blood (samples 5-8) were heated to 80° C. for 5minutes and then they were moved to a 55° C. heat block. The L-probe,wash buffer, streptavidin beads and proteinase K were added, and thereaction was incubated at 55° C. for 60 minutes. The samples wereremoved from the heat block and the magnetic beads were captured using adynal MPC 96S magnetic capture plate. The supernatant was removed andthe beads were washed 3 times with wash buffer. DyNamo SYBR green PCRmaster mix (25 ul, 1×) and universal primers (SEQ ID NOS 1 and 2, 300nM) were added to the beads and samples were heat cycled using aStratagene MX4000 realtime PCR instrument for 30 cycles (95° C. for 15minutes, 30 cycles 95° C. (10 s), 60° C(24 s), 72° C(10 s)). The Ctvalues were recorded and the amplified samples were injected into anAgilent Bioanalyzer 2100 so as to verify the length of the amplicons.All amplicons showed the correct size (˜96 bp) and the performance wascomparable for the blood and water samples demonstrating the ability todirectly analyze RNA in lysed blood. The results are summarizes in Table7 below.

TABLE 5 CLPA Probe Sets. Sample Identifier L-Probe (1 nM) S-Probe (1 nM)RNA Transcript 1-8 DDB2 SEQ ID NO: SEQ ID NO: Origene Plasmid 10 11SC122547

TABLE 6 DDB2 reaction mixture. Samples 1-4 5-8 GuHCL Lysis Buffer (2X)12.5 μl 12.5 μl S-Probe (5 nM) 1 μl 1 μl RNA Transcript (10 ng/ul to 1μl 1 μl 0.01 ng/ul) Whole Blood 0 μl 12.5 μl Water 12.5 ul 0 μl Heat 80°C. for 5 min, chill on ice Wash Buffer 20 μl 15 μl L-Probe (5 nM) 1 μl 1μl Dynal M-270 Beads 2 μl 2 μl Proteinase K (10 mg/ml) 0 μl 5 μl Total50 μl 50 μl Incubate 55° C. for 60 min. a) GuHCL lysis buffer (1X) is 3MGUHCL, 20 mM EDTA, 5 mM DTT, 1.5% Triton, 30 mM Tris pH 7.2). b) WashBuffer is 100 mM Tris (pH 7.4), 0.01% Triton.

TABLE 7 Summary results of water versus blood Assay DDB2 Conc Ct valueSample 1 10 ng 13.5 Water 2 1 ng 17 Water 3 0.1 ng 20.2 Water 4 0.01 ng24 Water 5 10 ng 13.5 Blood 6 1 ng 16 Blood 7 0.1 ng 19.2 Blood 8 0.01ng 23.5 Blood

EXAMPLE 4 3-probe CLPA-CE assay

Reactions were prepared in duplicate as presented in Table 8 using DNAtarget mimic probe SEQ ID NO 23 and the 3-probe CLPA probe set (SEQ IDNOS 20, 21 and 22). The probe numbers refer to the SEQ ID NOS inTable 1. The reagents were added in the concentrations and volumes inTable 9. The S-probes, L-probe and target mimics were heated to 50° C.for 60 minutes in a 0.2 mL PCR tube, after which 2.5 μl of the CLPAreaction was used as template in a Dynamo SYBR green PCR reaction with25 amplification cycles. Real-time PCR data was averaged for theduplicate samples and is presented in Table 8 (Ct value column). A 1 μlsample of each reaction was then analyzed via Agilent Bioanalyzer 2100to determine the size of the reaction product.

TABLE 8 CLPA Probe Sets. 3′-S 2L- 5′-S Target probe Probe Probe MimicAmpli- Sam- Identi- SEQ SEQ SEQ SEQ con Ct ples fier ID NO ID NO ID NOID NO size value 1&2 GAPDH 20 21 22 23 About 16.3 135 bp 3&4 Negative 2021 22 23 None No ob- CT served Probes at 1 nM concentration; targetmimic at 10 pM concentration.

TABLE 9 Reagent table-Example 1 1X PCR Buffer Buffer* 12.5 μl  3 and 5′S-Probe (10 nM) & 2L-Probe (10 nM) 2.5 μl each Target Mimic (1 nM) 2.5μl Water 2.5 μl Heat at 50 C. for 1 hour *1X PCR buffer is 1.5 mM MgCL2,50 mM KCl, 10 mM Tris-HCl pH 8.3

EXAMPLE 5 Multiplex Real-Time CLPA Detection of mRNA

In a 0.2 ml PCR tube was added 4 sets of CLPA reagents that wereengineered to possess unique binding sites for different color duallabeled probes. The reactions were prepared as indicated in Table 10 andTable 11. The CLPA probes sets and dual labeled probes correspond to SEQID NOS 25 through 36 in Table 1. The S and run-off transcript mRNA(GAPDH, PCNA and DDB2) were added to 2× lysis buffer (GuHCL lysis buffer(1×) is 3M GUHCL, 20 mM EDTA, 5 mM DTT, 1.5% Triton, 30 mM Tris pH 7.2)and heated to 80° C. for 5 min. The samples were cooled on ice andstreptavidin coated magnetic beads (DYNAL M-270) and L-probe were added.The samples were heated at 50° C. for 1 hour. The magnetic beads werecaptured on a DYNAL MPC plate and washed twice with wash buffer. Thebeads were recaptured and dynamo PCR 1× mastermix was added with the 4different dual labeled probes and universal PCR primers (25 ul totalvolume). The samples were heat cycled using a Stratagene MX4000 realtimePCR instrument for 30 cycles (95° C. for 15 minutes, 30 cycles 95° C.(10 s), 60° C. (24 s), 72° C. (10 s)) with proper filters for monitoringthe fluorescence in the FAM, Cal Fluor orange 560, Cal Fluor Red 610,and Quasar 670 channels. The Ct values observed for each channel wererecorded and are indicated in Table 10.

TABLE 10 Multiplex reagents used in Example 5. S Probes L Probes (25 pM)(25 pM) Ct(FAM)- Ct(560)- Ct(610)- Ct(670)- Samples SEQ ID NOs SEQ IDNOs Targets GAPDH3 DDB2 PCNA GAPDH4 1 & 2 26, 29, 25, 28, 250 ng yeasttRNA; 25.5 24.5 24.8 25.8 32, 35 31, 34 40 pg GAPDH(Origene SC118869),40 pg PCNA (SC118528), 40 pg DDB2 (SC122547) mRNA 3 & 4 26, 29, 25, 28,250 ng yeast tRNA No Ct No Ct No Ct No Ct 32, 35 31, 34 (negative) 5 & 626, 29, 25, 28, 250 ng yeast tRNA; 22.1 24.5 22.1 22.2 32, 35 31, 34 40pg GAPDH(Origene SC118869), 40 pg PCNA (SC118528), 40 pg DDB2 (SC122547)mRNA 7 & 8 26, 29, 25, 28, 250 ng yeast tRNA No Ct No Ct No Ct No Ct 32,35 31, 34 (negative)

TABLE 11 Additional reagents used in Example 5. GuHCL Lysis Buffer (2X)12.5 μl S-Probes (0.25 nM Stock of each) 5 μl mRNAs (250 ng tRNA +/−mRNAs) 5 μl Water 2.5 μl Heat 80° C. for 5 min, chill on ice Water 18 μlL-Probes (0.25 nm stock of each) 5 μl Beads 2 μl Total 50 μl Incubate50° C. 1 Hour

1. A method for detecting in a sample, comprising a plurality of samplenucleic acids of different sequence, the presence of at least onespecific target nucleic acid sequence comprising a first and a secondtarget domain, the domains located essentially adjacent to one another,comprising the steps of: a) contacting the sample nucleic acids with aplurality of different probes sets, each probe set comprising i. a firstligation probe comprising: 1) a first probe domain substantiallycomplementary to said first target domain; and 2) a firstnon-complementary region being essentially non-complementary to the saidtarget nucleic acid 3) a 5′-ligation moiety; and ii. second ligationprobe comprising: 1) a second probe domain substantially complementaryto said second target domain; 2) a second non-complementary region,being essentially non-complementary to the said target nucleic acid 3) a3′ ligation moiety; wherein at least one of said ligation probecomprises a variable spacer sequence; and b) ligating said first andsecond ligation probes in the absence of a ligase enzyme to form aligation product; c) amplifying said ligation product; and d) detectingthe presence of said ligation product.
 2. A method of claim 1 whereinsaid target sequence is RNA and/or DNA.
 3. A method of claim 1 whereinsaid target sequence comprises unpurified RNA
 4. The method of claim 1wherein said sample is derived from a mammalian body selected from thegroup consisting of blood, urine, saliva and feces. 5.-7. (canceled) 8.A method as in claim 1 wherein said step of detection is by massspectrometry.
 9. The method of claim 1 wherein said 5′ ligation moietyon said first ligation probe is DAB SYL moiety and said 3′ ligationmoiety on said second ligation probe is 3-phophorothioate moiety.
 10. Amethod as in claim 1 wherein said first and second ligation probes eachfurther comprising a universal primer sequence for amplification of saidligation product.
 11. The method of claim 10 wherein one of theuniversal primers that binds said primer sequence contains a detectablelabel.
 12. (canceled)
 13. (canceled)
 14. A method for detecting in asample, comprising a plurality of sample nucleic acids of differentsequence, the presence of at least one specific target nucleic acidsequence comprising a first and a second target domain, the domainslocated essentially adjacent to one another, comprising the steps of: a)Contacting the sample nucleic acids with a plurality of different probessets, each probe set comprising i) a first ligation probe comprising: 1)a first probe domain substantially complementary to said first targetdomain; and 2) a first non-complementary region being essentiallynon-complementary to the said target nucleic acid 3) a 5′-ligationmoiety; and ii) second ligation probe comprising: 1) a second probedomain substantially complementary to said second target domain; 2) asecond non-complementary region, being essentially non-complementary tothe said target nucleic acid; and 3) a 3′ ligation moiety; wherein atleast one of said first and second ligation probes further comprises ananchor sequence b) capturing said ligation product on a microarraysubstrate comprising a capture probe substantially complementary to saidanchor sequence; and c) detecting the presence of said ligated product.15. A method as in claim 14 wherein said first and second ligationprobes each further comprising a universal primer sequence foramplification of said ligation product wherein one of the universalprimers that binds said primer sequence contains a detectable labelwherein said detectable label is selected from the group consisting of afluorescent label, an electrochemical label and a magnetic label.
 16. Amethod for detecting in a sample, comprising a plurality of samplenucleic acids of different sequence, the presence of at least onespecific target nucleic acid sequence comprising a first and a secondtarget domain, and a third domain located between the first and seconddomains, the domains located essentially adjacent to one another,comprising the steps of: b) contacting the sample nucleic acids with aplurality of different probes sets, each probe set comprising i. a firstligation probe comprising: 1) a first probe domain substantiallycomplementary to said first target domain; and 2) a firstnon-complementary region being essentially non-complementary to the saidtarget nucleic acid 3) a 5′-ligation moiety; and ii. second ligationprobe comprising: 1) a second probe domain substantially complementaryto said second target domain; 2) a second non-complementary region,being essentially non-complementary to the said target nucleic acid 3) a3′ ligation moiety; iii. a third ligation probe comprising 1) a thirdprobe domain substantially complementary to the said third target domain2) a 3′ and a 5′ ligation moiety; b) ligating said first ligation probe,said second ligation probe, and said third ligation probe in the absenceof a ligase enzyme to form a ligation product; c) amplifying saidligation product; and c) detecting the presence of said ligated product.17. A method of claim 16 wherein said target sequence is RNA and/or DNA.18. The method of claim 16 wherein said sample is derived from amammalian body selected from the group consisting of blood, urine,saliva and feces. 19.22. (canceled)
 23. A method as in claim 16 whereinsaid first and second ligation probes each further comprising auniversal primer sequence for amplification of said ligation product.24. The method of claim 16 wherein one of the universal primers thatbinds said primer sequence contains a detectable label. 25.-31.(canceled)